Engineered skin equivalent, method of manufacture thereof and products derived therefrom

ABSTRACT

Disclosed herein are synthetic leathers, artificial epidermal layers, artificial dermal layers, layered structures, products produced therefrom and methods of producing the same.

CROSS REFERENCE

This application claims priority to U.S. Provisional Application No.62/325,819, filed on Apr. 21, 2016, which is herein incorporated byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with the support of National Institutes ofHealth (NIH) Grant Number R21 AR061583 and RO1 AR051930, MedicalResearch Council (UK) Grant Number G0801061, Research Service of theDepartment of Veterans Affairs and Dystrophic Epidermolysis BullosaResearch Association.

SUMMARY OF THE DISCLOSURE

Disclosed herein are methods of making a synthetic leather. In someembodiments, the method can comprise forming an artificial dermal layercomprising a fibroblast. In some embodiments, the method can comprisetanning at least a portion of a dermal layer, thereby forming asynthetic leather. In some embodiments, a fibroblast can bedifferentiated from an induced pluripotent stem cell. In someembodiments, the method can further comprise forming an artificialepidermal layer. In some embodiments, an epidermal layer can comprise akeratinocyte. In some embodiments, the method can comprise placing anepidermal layer upon a dermal layer thereby forming a layered structure.In some embodiments, a keratinocyte can be a mammalian keratinocyte. Insome embodiments, a mammal can be a non-human mammal. In someembodiments, a keratinocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a keratinocyte can expressKRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or acombination thereof. In some embodiments, a layered structure canfurther comprise a melanocyte. In some embodiments, a melanocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a melanocyte can express Sox-10, MITF-M, gp-100, DCT, TYR,MLANA or a combination thereof. In some embodiments, a synthetic leathercan comprise a pigment. In some embodiments, a melanocyte can be amammalian melanocyte. In some embodiments, a mammal can be a human. Insome embodiments, a fibroblast can express CD10, CD73, CD44, CD90, typeI collagen, type III collagen, prolyl-4-hydroxylase beta, or acombination thereof. In some embodiments, a fibroblast can be amammalian fibroblast. In some embodiments, a mammal can be a non-humanmammal. In some embodiments, a non-human mammal can be one of a primate,bovine, ovine, porcine, equinine, canine, feline, rodent, or lagomorph.In some embodiments, a fibroblast can be a non-mammalian fibroblast. Insome embodiments, a non-mammal can be a fish, a bird or a reptile. Insome embodiments, an epidermal layer can further comprise collagen. Insome embodiments, an epidermal layer can be subjected to furtherprocessing. In some embodiments, a layered structure can furthercomprise collagen. In some embodiments, a layered structure can besubjected to further processing. In some embodiments, a dermal layer canfurther comprise collagen. In some embodiments, a dermal layer can besubjected to further processing. In some embodiments, processing can beselected from a group consisting of preserving, soaking, bating,pickling, depickling, thinning, ramming, lubricating, crusting, wetting,sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring,filling, stripping, stuffing, whitening, fixating, setting, drying,conditioning, milling, staking, buffing, finishing, oiling, brushing,padding, impregnating, spraying, roller coating, curtain coating,polishing, plating, embossing, ironing, glazing, tumbling and anycombination thereof. In some embodiments, collagen can be produced atleast in part by a collagen producing cell, can be separately added, orany combination thereof. In some embodiments, a collagen producing cellcan comprise an epithelial cell, a keratinocyte, a fibroblast, acomeocyte, a melanocyte, a Langerhans cell, a basal cell, or acombination thereof. In some embodiments, a collagen producing cell cancomprise a epithelial cell wherein the epithelial cell can comprise asquamous cell, a cuboidal cell, a columnar cell, a basal cell, or acombination thereof. In some embodiments, a collagen producing cell cancomprise a keratinocyte wherein the keratinocyte can comprise anepithelial keratinocyte, basal keratinocyte, proliferating basalkeratinocyte, differentiated suprabasal keratinocyte, or a combinationthereof. In some embodiments, a collagen producing cell can comprise asmooth muscle cell. In some embodiments, a synthetic leather can furthercomprise one or more of keratin, elastin, gelatin, proteoglycan,dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin,dermatopontin, lipid, fatty acid, carbohydrate, or a combinationthereof. In some embodiments, a thickness of a dermal layer can rangefrom about 0.02 mm to about 5 mm. In some embodiments, a thickness of adermal layer can range from about 0.1 mm to about 0.5 mm. In someembodiments, a synthetic leather can further comprise a first dermallayer and a second dermal layer. In some embodiments, a first dermallayer can be placed upon a second dermal layer. In some embodiments, athickness of an epidermal layer can range from about 0.01 mm to about 2mm. In some embodiments, a thickness of an epidermal layer can rangefrom about 0.1 mm to about 0.2 mm. In some embodiments, a syntheticleather can further comprise a first epidermal layer and a secondepidermal layer. In some embodiments, a synthetic leather can furthercomprise a basement membrane substitute. In some embodiments, a basementmembrane substitute can be between an epidermal layer and a dermallayer. In some embodiments, a basement membrane substitute can comprisea dried acellular amniotic membrane. In some embodiments, a dermal layercan be formed upon a scaffold. In some embodiments, a scaffold can benatural or synthetic. In some embodiments, a scaffold can comprise silk.In some embodiments, a scaffold can comprise chitosan. In someembodiments, a scaffold can comprise a natural tissue adhesive. In someembodiments, a natural tissue adhesive can comprise fibrin glue. In someembodiments, a scaffold can be comprised in part in a synthetic leather.In some embodiments, a dermal layer or an epidermal layer can becultured in vitro. In some embodiments, a dermal layer can be culturedin vitro. In some embodiments, a dermal layer can be cultured with asupplement. In some embodiments, a supplement can comprise one or moreof collagen, fibrin, growth factors, ascorbic acid, dextran sulphate, orcarrageenan. In some embodiments, a supplement can be a naturalsupplement. In some embodiments, a supplement can be a syntheticsupplement. In some embodiments, an induced pluripotent stem cell can beproduced through the induced gene expression of Oct3, Oct4, Sox2, Klf4,c-Myc or a combination thereof in an adult somatic cell. In someembodiments, at least a portion of a leather article can be formed fromthe methods disclosed herein. In some embodiments, a leather article cancomprise one or more of a watch strap, a belt, a packaging, a shoe, aboot, a footwear, a glove, a clothing, a luggage, a bag, a clutch, apurse, a backpack, a wallet, a saddle, a harness, a whip, an interior,an exterior, an upholstery, a book binding, a furniture, a lamp, a lampshade, a table covering, a wall covering, a floor covering, a ceilingcovering, a car interior, a car exterior, a boat interior, a boatexterior, an airplane interior, a yacht interior, a yacht exterior, apillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a leather article can be a watch strap. In someembodiments, a leather article can be a belt. In some embodiments, aleather article can be a bag. At least about 2% of cells comprised in asynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in synthetic leather can bedifferentiated from an induced pluripotent stem cell. At least about 50%of cells comprised in a synthetic leather can be differentiated from aninduced pluripotent stem cell.

Disclosed herein are methods of making a synthetic leather. In someembodiments, the method can comprise placing an artificial epidermallayer upon an artificial dermal layer thereby forming a layeredstructure. In some embodiments, an epidermal layer can comprise akeratinocyte and a dermal layer can comprise a fibroblast. In someembodiments, the method can comprise tanning at least a portion of alayered structure, thereby forming a synthetic leather. In someembodiments, a fibroblast or a keratinocyte can be differentiated froman induced pluripotent stem cell. In some embodiments, a keratinocytecan be a mammalian keratinocyte. In some embodiments, a mammal can be anon-human mammal. In some embodiments, a keratinocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a keratinocyte can express KRT14, p63, DSG3, ITGB4, LAMA5,KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments,a layered structure can further comprise a melanocyte. In someembodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a synthetic leather can comprise a pigment. In someembodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, a mammal can be a human. In some embodiments, a fibroblastcan be differentiated from an induced pluripotent stem cell. In someembodiments, a fibroblast can express CD10, CD73, CD44, CD90, type Icollagen, type III collagen, prolyl-4-hydroxylase beta, or a combinationthereof. In some embodiments, a fibroblast can be a mammalianfibroblast. In some embodiments, a mammal can be a non-human mammal. Insome embodiments, a non-human mammal can be one of a primate, bovine,ovine, porcine, equinine, canine, feline, rodent, or lagomorph. In someembodiments, a fibroblast can be a non-mammalian fibroblast. In someembodiments, a non-mammal can be a fish, a bird or a reptile. In someembodiments, an epidermal layer can further comprise collagen. In someembodiments, an epidermal layer can be subjected to further processing.In some embodiments, a layered structure can further comprise collagen.In some embodiments, a layered structure can be subjected to furtherprocessing. In some embodiments, a dermal layer can further comprisecollagen. In some embodiments, a dermal layer can be subjected tofurther processing. In some embodiments, processing can be selected froma group consisting of preserving, soaking, bating, pickling, depickling,thinning, retanning, lubricating, crusting, wetting, sammying, shaving,rechroming, neutralizing, dyeing, fatliquoring, filling, stripping,stuffing, whitening, fixating, setting, drying, conditioning, milling,staking, buffing, finishing, oiling, brushing, padding, impregnating,spraying, roller coating, curtain coating, polishing, plating,embossing, ironing, glazing, tumbling and any combination thereof. Insome embodiments, collagen can be produced at least in part by acollagen producing cell, can be separately added, or any combinationthereof. In some embodiments, a collagen producing cell can comprise anepithelial cell, a keratinocyte, a fibroblast, a comeocyte, amelanocyte, a Langerhans cell, a basal cell, or a combination thereof.In some embodiments, a collagen producing cell can comprise a epithelialcell wherein the epithelial cell can comprise a squamous cell, acuboidal cell, a columnar cell, a basal cell, or a combination thereof.In some embodiments, a collagen producing cell can comprise akeratinocyte wherein the keratinocyte can comprise an epithelialkeratinocyte, basal keratinocyte, proliferating basal keratinocyte,differentiated suprabasal keratinocyte, or a combination thereof. Insome embodiments, a collagen producing cell can comprise a smooth musclecell. In some embodiments, a synthetic leather can further comprise oneor more of keratin, elastin, gelatin, proteoglycan, dermatan sulfateproteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin,lipid, fatty acid, carbohydrate, or a combination thereof. In someembodiments, a thickness of a dermal layer can range from about 0.02 mmto about 5 mm. In some embodiments, a thickness of a dermal layer canrange from about 0.1 mm to about 0.5 mm. In some embodiments, asynthetic leather can further comprise a first dermal layer and a seconddermal layer. In some embodiments, a first dermal layer can be placedupon a second dermal layer. In some embodiments, a thickness of anepidermal layer can range from about 0.01 mm to about 2 mm. In someembodiments, a thickness of an epidermal layer can range from about 0.1mm to about 0.2 mm. In some embodiments, a synthetic leather can furthercomprise a first epidermal layer and a second epidermal layer. In someembodiments, a synthetic leather can further comprise a basementmembrane substitute. In some embodiments, a basement membrane substitutecan be between an epidermal layer and a dermal layer. In someembodiments, a basement membrane substitute can comprise a driedacellular amniotic membrane. In some embodiments, a dermal layer can beformed upon a scaffold. In some embodiments, a scaffold can be naturalor synthetic. In some embodiments, a scaffold can comprise silk. In someembodiments, a scaffold can comprise chitosan. In some embodiments, ascaffold can comprise a natural tissue adhesive. In some embodiments, anatural tissue adhesive can comprise fibrin glue. In some embodiments, ascaffold can be comprised in part in a synthetic leather. In someembodiments, a dermal layer or an epidermal layer can be cultured invitro. In some embodiments, a dermal layer can be cultured in vitro. Insome embodiments, a dermal layer can be cultured with a supplement. Insome embodiments, a supplement can comprise one or more of collagen,fibrin, growth factors, ascorbic acid, dextran sulphate, or carrageenan.In some embodiments, a supplement can be a natural supplement. In someembodiments, a supplement can be a synthetic supplement. In someembodiments, an induced pluripotent stem cell can be produced throughthe induced gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc or acombination thereof in an adult somatic cell. In some embodiments, atleast a portion of a leather article can be formed from the methodsdisclosed herein. In some embodiments, a leather article can compriseone or more of a watch strap, a belt, a packaging, a shoe, a boot, afootwear, a glove, a clothing, a luggage, a bag, a clutch, a purse, abackpack, a wallet, a saddle, a harness, a whip, an interior, anexterior, an upholstery, a book binding, a furniture, a lamp, a lampshade, a table covering, a wall covering, a floor covering, a ceilingcovering, a car interior, a car exterior, a boat interior, a boatexterior, an airplane interior, a yacht interior, a yacht exterior, apillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a leather article can be a watch strap. In someembodiments, a leather article can be a belt. In some embodiments, aleather article can be a bag. At least about 2% of cells comprised in asynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in synthetic leather can bedifferentiated from an induced pluripotent stem cell. At least about 50%of cells comprised in a synthetic leather can be differentiated from aninduced pluripotent stem cell.

Disclosed herein are methods of making a synthetic leather. In someembodiments, the method can comprise placing an artificial epidermallayer upon an artificial dermal layer thereby forming a layeredstructure. In some embodiments, an epidermal layer can comprise akeratinocyte and a dermal layer can comprise a fibroblast. In someembodiments, the method can comprise removing at least a portion of anepidermal layer from a layered structure to form a removed product. Insome embodiments, the method can comprise tanning at least a portion ofa removed product, thereby forming a synthetic leather. In someembodiments, a fibroblast or a keratinocyte can be differentiated froman induced pluripotent stem cell. In some embodiments, a keratinocytecan be a mammalian keratinocyte. In some embodiments, a mammal can be anon-human mammal. In some embodiments, a keratinocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a keratinocyte can express KRT14, p63, DSG3, ITGB4, LAMA5,KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments,a layered structure can further comprise a melanocyte. In someembodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a synthetic leather can comprise a pigment. In someembodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, a mammal can be a human. In some embodiments, a fibroblastcan be differentiated from an induced pluripotent stem cell. In someembodiments, a fibroblast can express CD10, CD73, CD44, CD90, type Icollagen, type III collagen, prolyl-4-hydroxylase beta, or a combinationthereof. In some embodiments, a removed product can further comprisecollagen. In some embodiments, a removed product can be subjected tofurther processing. In some embodiments, a fibroblast can be a mammalianfibroblast. In some embodiments, a mammal can be a non-human mammal. Insome embodiments, a non-human mammal can be one of a primate, bovine,ovine, porcine, equinine, canine, feline, rodent, or lagomorph. In someembodiments, a fibroblast can be a non-mammalian fibroblast. In someembodiments, a non-mammal can be a fish, a bird or a reptile. In someembodiments, an epidermal layer can further comprise collagen. In someembodiments, an epidermal layer can be subjected to further processing.In some embodiments, a layered structure can further comprise collagen.In some embodiments, a layered structure can be subjected to furtherprocessing. In some embodiments, a dermal layer can further comprisecollagen. In some embodiments, a dermal layer can be subjected tofurther processing. In some embodiments, processing can be selected froma group consisting of preserving, soaking, bating, pickling, depickling,thinning, ramming, lubricating, crusting, wetting, sammying, shaving,rechroming, neutralizing, dyeing, fatliquoring, filling, stripping,stuffing, whitening, fixating, setting, drying, conditioning, milling,staking, buffing, finishing, oiling, brushing, padding, impregnating,spraying, roller coating, curtain coating, polishing, plating,embossing, ironing, glazing, tumbling and any combination thereof. Insome embodiments, collagen can be produced at least in part by acollagen producing cell, can be separately added, or any combinationthereof. In some embodiments, a collagen producing cell can comprise anepithelial cell, a keratinocyte, a fibroblast, a comeocyte, amelanocyte, a Langerhans cell, a basal cell, or a combination thereof.In some embodiments, a collagen producing cell can comprise a epithelialcell wherein the epithelial cell can comprise a squamous cell, acuboidal cell, a columnar cell, a basal cell, or a combination thereof.In some embodiments, a collagen producing cell can comprise akeratinocyte wherein the keratinocyte can comprise an epithelialkeratinocyte, basal keratinocyte, proliferating basal keratinocyte,differentiated suprabasal keratinocyte, or a combination thereof. Insome embodiments, a collagen producing cell can comprise a smooth musclecell. In some embodiments, a synthetic leather can further comprise oneor more of keratin, elastin, gelatin, proteoglycan, dermatan sulfateproteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin,lipid, fatty acid, carbohydrate, or a combination thereof. In someembodiments, a thickness of a dermal layer can range from about 0.02 mmto about 5 mm. In some embodiments, a thickness of a dermal layer canrange from about 0.1 mm to about 0.5 mm. In some embodiments, asynthetic leather can further comprise a first dermal layer and a seconddermal layer. In some embodiments, a first dermal layer can be placedupon a second dermal layer. In some embodiments, a thickness of anepidermal layer can range from about 0.01 mm to about 2 mm. In someembodiments, a thickness of an epidermal layer can range from about 0.1mm to about 0.2 mm. In some embodiments, a synthetic leather can furthercomprise a first epidermal layer and a second epidermal layer. In someembodiments, a synthetic leather can further comprise a basementmembrane substitute. In some embodiments, a basement membrane substitutecan be between an epidermal layer and a dermal layer. In someembodiments, a basement membrane substitute can comprise a driedacellular amniotic membrane. In some embodiments, a dermal layer can beformed upon a scaffold. In some embodiments, a scaffold can be naturalor synthetic. In some embodiments, a scaffold can comprise silk. In someembodiments, a scaffold can comprise chitosan. In some embodiments, ascaffold can comprise a natural tissue adhesive. In some embodiments, anatural tissue adhesive can comprise fibrin glue. In some embodiments, ascaffold can be comprised in part in a synthetic leather. In someembodiments, a dermal layer or an epidermal layer can be cultured invitro. In some embodiments, a dermal layer can be cultured in vitro. Insome embodiments, a dermal layer can be cultured with a supplement. Insome embodiments, a supplement can comprise one or more of collagen,fibrin, growth factors, ascorbic acid, dextran sulphate, or carrageenan.In some embodiments, a supplement can be a natural supplement. In someembodiments, a supplement can be a synthetic supplement. In someembodiments, an induced pluripotent stem cell can be produced throughthe induced gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc or acombination thereof in an adult somatic cell. In some embodiments, atleast a portion of a leather article can be formed from the methodsdisclosed herein. In some embodiments, a leather article can compriseone or more of a watch strap, a belt, a packaging, a shoe, a boot, afootwear, a glove, a clothing, a luggage, a bag, a clutch, a purse, abackpack, a wallet, a saddle, a harness, a whip, an interior, anexterior, an upholstery, a book binding, a furniture, a lamp, a lampshade, a table covering, a wall covering, a floor covering, a ceilingcovering, a car interior, a car exterior, a boat interior, a boatexterior, an airplane interior, a yacht interior, a yacht exterior, apillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a leather article can be a watch strap. In someembodiments, a leather article can be a belt. In some embodiments, aleather article can be a bag. At least about 2% of cells comprised in asynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in synthetic leather can bedifferentiated from an induced pluripotent stem cell. At least about 50%of cells comprised in a synthetic leather can be differentiated from aninduced pluripotent stem cell.

Disclosed herein are tanned synthetic leathers. In some embodiments,prior to tanning a tanned synthetic leather can comprise an artificialdermal layer comprising fibroblast. In some embodiments, a fibroblastcan be differentiated from an induced pluripotent stem cell. In someembodiments, prior to tanning, a tanned synthetic leather can furthercomprise an artificial epidermal layer. In some embodiments, anepidermal layer can further comprise a keratinocyte. In someembodiments, an epidermal layer can be upon a dermal layer therebyforming a layered structure. In some embodiments, a keratinocyte can bea mammalian keratinocyte. In some embodiments, a mammal can be anon-human mammal. In some embodiments, a keratinocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a keratinocyte can express KRT14, p63, DSG3, ITGB4, LAMA5,KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments,a layered structure can further comprise a melanocyte. In someembodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a synthetic leather can further comprise a pigment. Insome embodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, a mammal can be a non-human mammal. In some embodiments, afibroblast can express CD10, CD73, CD44, CD90, type I collagen, type IIIcollagen, prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, a fibroblast can be a mammalian fibroblast. In someembodiments, a mammal can be a non-human mammal. In some embodiments, anon-human mammal can be one of a primate, bovine, ovine, porcine,equinine, canine, feline, rodent, or lagomorph. In some embodiments, afibroblast can be a non-mammalian fibroblast. In some embodiments, anon-mammal can be a fish, a bird or a reptile. In some embodiments, anepidermal layer cam further comprises collagen. In some embodiments, alayered structure can further comprise collagen. In some embodiments, adermal layer can further comprise collagen. In some embodiments,collagen can be produced at least in part by a collagen producing cell,can be separately added, or any combination thereof. In someembodiments, a collagen producing cell can comprise an epithelial cell,a keratinocyte, a fibroblast, a comeocyte, a melanocyte, a Langerhanscell, a basal cell, or a combination thereof. In some embodiments, acollagen producing cell can comprise an epithelial cell wherein anepithelial cell can comprise a squamous cell, a cuboidal cell, acolumnar cell, a basal cell, or a combination thereof. In someembodiments, a collagen producing cell can comprises a keratinocytewherein a keratinocyte comprises epithelial keratinocyte, basalkeratinocyte, proliferating basal keratinocyte, differentiatedsuprabasal keratinocyte, or a combination thereof. In some embodiments,a collagen producing cells can comprise a smooth muscle cell. In someembodiments, a synthetic leather can further comprise one or more ofkeratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan,glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fattyacid, carbohydrate, or a combination thereof. In some embodiments, athickness of a dermal layer can range from about 0.02 mm to about 5 mm.In some embodiments, a thickness of a dermal layer can range from about0.1 mm to about 0.5 mm. In some embodiments, a synthetic leather canfurther comprise a first dermal layer and a second dermal layer. In someembodiments, a first dermal layer can be upon a second dermal layer. Insome embodiments, a thickness of an epidermal layer can range from about0.01 mm to about 2 mm. In some embodiments, a thickness of an epidermallayer can range from about 0.1 mm to about 0.2 mm. In some embodiments,a synthetic leather can further comprise a first epidermal layer and asecond epidermal layer. In some embodiments, a synthetic leather cancomprise a basement membrane substitute. In some embodiments, a basementmembrane substitute can be between an epidermal layer and an dermallayer. In some embodiments, a basement membrane substitute can comprisea dried acellular amniotic membrane. In some embodiments, a dermal layercan be formed upon a scaffold. In some embodiments, a scaffold can benatural or synthetic. In some embodiments, a scaffold can comprise silk.In some embodiments, a scaffold can comprise chitosan. In someembodiments, a scaffold can comprise a natural tissue adhesive. In someembodiments, a natural tissue adhesive can comprise fibrin glue. In someembodiments, a scaffold can be comprised in part in a tanned syntheticleather. In some embodiments, a dermal layer or an epidermal layer canbe cultured in vitro. In some embodiments, a dermal layer can becultured in vitro. In some embodiments, a tanned synthetic leather canbe comprised in one or more of a watch strap, a belt, a packaging, ashoe, a boot, a footwear, a glove, a clothing, a luggage, a bag, aclutch, a purse, a backpack, a wallet, a saddle, a harness, a whip, aninterior, an exterior, an upholstery, a book binding, a furniture, alamp, a lamp shade, a table covering, a wall covering, a floor covering,a ceiling covering, a car interior, a car exterior, a boat interior, aboat exterior, an airplane interior, a yacht interior, a yacht exterior,a pillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a tanned synthetic leather can be comprised in a watchstrap. In some embodiments, a tanned synthetic leather can be comprisedin a belt. In some embodiments, a tanned synthetic leather can becomprised in a bag. At least about 2% of cells comprised in a tannedsynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in tanned synthetic leathercan be differentiated from an induced pluripotent stem cell. At leastabout 50% of cells comprised in a tanned synthetic leather can bedifferentiated from an induced pluripotent stem cell.

Disclosed herein are tanned synthetic leathers. In some embodiments,prior to tanning a tanned synthetic leather can comprise a layeredstructure. In some embodiments, a layered structure can comprise anartificial dermal layer. In some embodiments, a dermal layer cancomprise a fibroblast. In some embodiments, a layered structure cancomprise an artificial epidermal layer. In some embodiments an epidermallayer can comprise a keratinocyte. In some embodiments, a fibroblast ora keratinocyte can be differentiated from an induced pluripotent stemcell. In some embodiments, a fibroblast can be differentiated from aninduced pluripotent stem cell. In some embodiments, a keratinocyte canbe a mammalian keratinocyte. In some embodiments, a mammal can be anon-human mammal. In some embodiments, a keratinocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a keratinocyte can express KRT14, p63, DSG3, ITGB4, LAMA5,KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments,a layered structure can further comprise a melanocyte. In someembodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a synthetic leather can further comprise a pigment. Insome embodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, a mammal can be a non-human mammal. In some embodiments, afibroblast can express CD10, CD73, CD44, CD90, type I collagen, type IIIcollagen, prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, a fibroblast can be a mammalian fibroblast. In someembodiments, a mammal can be a non-human mammal. In some embodiments, anon-human mammal can be one of a primate, bovine, ovine, porcine,equinine, canine, feline, rodent, or lagomorph. In some embodiments, afibroblast can be a non-mammalian fibroblast. In some embodiments, anon-mammal can be a fish, a bird or a reptile. In some embodiments, anepidermal layer cam further comprises collagen. In some embodiments, alayered structure can further comprise collagen. In some embodiments, adermal layer can further comprise collagen. In some embodiments,collagen can be produced at least in part by a collagen producing cell,can be separately added, or any combination thereof. In someembodiments, a collagen producing cell can comprise an epithelial cell,a keratinocyte, a fibroblast, a comeocyte, a melanocyte, a Langerhanscell, a basal cell, or a combination thereof. In some embodiments, acollagen producing cell can comprise an epithelial cell wherein anepithelial cell can comprise a squamous cell, a cuboidal cell, acolumnar cell, a basal cell, or a combination thereof. In someembodiments, a collagen producing cell can comprises a keratinocytewherein a keratinocyte comprises epithelial keratinocyte, basalkeratinocyte, proliferating basal keratinocyte, differentiatedsuprabasal keratinocyte, or a combination thereof. In some embodiments,a collagen producing cells can comprise a smooth muscle cell. In someembodiments, a synthetic leather can further comprise one or more ofkeratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan,glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fattyacid, carbohydrate, or a combination thereof. In some embodiments, athickness of a dermal layer can range from about 0.02 mm to about 5 mm.In some embodiments, a thickness of a dermal layer can range from about0.1 mm to about 0.5 mm. In some embodiments, a synthetic leather canfurther comprise a first dermal layer and a second dermal layer. In someembodiments, a first dermal layer can be upon a second dermal layer. Insome embodiments, a thickness of an epidermal layer can range from about0.01 mm to about 2 mm. In some embodiments, a thickness of an epidermallayer can range from about 0.1 mm to about 0.2 mm. In some embodiments,a synthetic leather can further comprise a first epidermal layer and asecond epidermal layer. In some embodiments, a synthetic leather cancomprise a basement membrane substitute. In some embodiments, a basementmembrane substitute can be between an epidermal layer and an dermallayer. In some embodiments, a basement membrane substitute can comprisea dried acellular amniotic membrane. In some embodiments, a dermal layercan be formed upon a scaffold. In some embodiments, a scaffold can benatural or synthetic. In some embodiments, a scaffold can comprise silk.In some embodiments, a scaffold can comprise chitosan. In someembodiments, a scaffold can comprise a natural tissue adhesive. In someembodiments, a natural tissue adhesive can comprise fibrin glue. In someembodiments, a scaffold can be comprised in part in a tanned syntheticleather. In some embodiments, a dermal layer or an epidermal layer canbe cultured in vitro. In some embodiments, a dermal layer can becultured in vitro. In some embodiments, a tanned synthetic leather canbe comprised in one or more of a watch strap, a belt, a packaging, ashoe, a boot, a footwear, a glove, a clothing, a luggage, a bag, aclutch, a purse, a backpack, a wallet, a saddle, a harness, a whip, aninterior, an exterior, an upholstery, a book binding, a furniture, alamp, a lamp shade, a table covering, a wall covering, a floor covering,a ceiling covering, a car interior, a car exterior, a boat interior, aboat exterior, an airplane interior, a yacht interior, a yacht exterior,a pillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a tanned synthetic leather can be comprised in a watchstrap. In some embodiments, a tanned synthetic leather can be comprisedin a belt. In some embodiments, a tanned synthetic leather can becomprised in a bag. At least about 2% of cells comprised in a tannedsynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in tanned synthetic leathercan be differentiated from an induced pluripotent stem cell. At leastabout 50% of cells comprised in a tanned synthetic leather can bedifferentiated from an induced pluripotent stem cell.

Disclosed herein are tanned synthetic leathers. In some embodiments,prior to tanning a tanned synthetic leather can comprise a removedproduct comprising a layered structure. In some embodiments, a layeredstructure can comprise an artificial dermal layer. In some embodiments,a dermal layer can comprise a fibroblast. In some embodiments, a layeredstructure can comprise an artificial epidermal layer. In someembodiments, an epidermal layer can comprise a keratinocyte. In someembodiments, a portion of an epidermal layer can be removed. In someembodiments, a fibroblast or a keratinocyte can be differentiated froman induced pluripotent stem cell. In some embodiments, a removed productcan further comprise collagen. In some embodiments, a fibroblast can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a keratinocyte can be a mammalian keratinocyte. In someembodiments, a mammal can be a non-human mammal. In some embodiments, akeratinocyte can be differentiated from an induced pluripotent stemcell. In some embodiments, a keratinocyte can express KRT14, p63, DSG3,ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. Insome embodiments, a layered structure can further comprise a melanocyte.In some embodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a synthetic leather can further comprise a pigment. Insome embodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, a mammal can be a non-human mammal. In some embodiments, afibroblast can express CD10, CD73, CD44, CD90, type I collagen, type IIIcollagen, prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, a fibroblast can be a mammalian fibroblast. In someembodiments, a mammal can be a non-human mammal. In some embodiments, anon-human mammal can be one of a primate, bovine, ovine, porcine,equinine, canine, feline, rodent, or lagomorph. In some embodiments, afibroblast can be a non-mammalian fibroblast. In some embodiments, anon-mammal can be a fish, a bird or a reptile. In some embodiments, anepidermal layer cam further comprises collagen. In some embodiments, alayered structure can further comprise collagen. In some embodiments, adermal layer can further comprise collagen. In some embodiments,collagen can be produced at least in part by a collagen producing cell,can be separately added, or any combination thereof. In someembodiments, a collagen producing cell can comprise an epithelial cell,a keratinocyte, a fibroblast, a comeocyte, a melanocyte, a Langerhanscell, a basal cell, or a combination thereof. In some embodiments, acollagen producing cell can comprise an epithelial cell wherein anepithelial cell can comprise a squamous cell, a cuboidal cell, acolumnar cell, a basal cell, or a combination thereof. In someembodiments, a collagen producing cell can comprises a keratinocytewherein a keratinocyte comprises epithelial keratinocyte, basalkeratinocyte, proliferating basal keratinocyte, differentiatedsuprabasal keratinocyte, or a combination thereof. In some embodiments,a collagen producing cells can comprise a smooth muscle cell. In someembodiments, a synthetic leather can further comprise one or more ofkeratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan,glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fattyacid, carbohydrate, or a combination thereof. In some embodiments, athickness of a dermal layer can range from about 0.02 mm to about 5 mm.In some embodiments, a thickness of a dermal layer can range from about0.1 mm to about 0.5 mm. In some embodiments, a synthetic leather canfurther comprise a first dermal layer and a second dermal layer. In someembodiments, a first dermal layer can be upon a second dermal layer. Insome embodiments, a thickness of an epidermal layer can range from about0.01 mm to about 2 mm. In some embodiments, a thickness of an epidermallayer can range from about 0.1 mm to about 0.2 mm. In some embodiments,a synthetic leather can further comprise a first epidermal layer and asecond epidermal layer. In some embodiments, a synthetic leather cancomprise a basement membrane substitute. In some embodiments, a basementmembrane substitute can be between an epidermal layer and an dermallayer. In some embodiments, a basement membrane substitute can comprisea dried acellular amniotic membrane. In some embodiments, a dermal layercan be formed upon a scaffold. In some embodiments, a scaffold can benatural or synthetic. In some embodiments, a scaffold can comprise silk.In some embodiments, a scaffold can comprise chitosan. In someembodiments, a scaffold can comprise a natural tissue adhesive. In someembodiments, a natural tissue adhesive can comprise fibrin glue. In someembodiments, a scaffold can be comprised in part in a tanned syntheticleather. In some embodiments, a dermal layer or an epidermal layer canbe cultured in vitro. In some embodiments, a dermal layer can becultured in vitro. In some embodiments, a tanned synthetic leather canbe comprised in one or more of a watch strap, a belt, a packaging, ashoe, a boot, a footwear, a glove, a clothing, a luggage, a bag, aclutch, a purse, a backpack, a wallet, a saddle, a harness, a whip, aninterior, an exterior, an upholstery, a book binding, a furniture, alamp, a lamp shade, a table covering, a wall covering, a floor covering,a ceiling covering, a car interior, a car exterior, a boat interior, aboat exterior, an airplane interior, a yacht interior, a yacht exterior,a pillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. In someembodiments, a tanned synthetic leather can be comprised in a watchstrap. In some embodiments, a tanned synthetic leather can be comprisedin a belt. In some embodiments, a tanned synthetic leather can becomprised in a bag. At least about 2% of cells comprised in a tannedsynthetic leather can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in tanned synthetic leathercan be differentiated from an induced pluripotent stem cell. At leastabout 50% of cells comprised in a tanned synthetic leather can bedifferentiated from an induced pluripotent stem cell.

Disclosed herein are artificial epidermal layers. In some embodiments,an artificial epidermal layer can comprise a hair follicle cell and amelanocyte. In some embodiments, an artificial epidermal layer cancomprise a hair follicle cell. In some embodiments, an artificialepidermal layer can comprise a melanocyte. In some embodiments, amelanocyte can be differentiated from an induced pluripotent stem cell.In some embodiments, a hair follicle cell can comprises a dermal papillacell, an outer root sheath cell or a combination thereof. In someembodiments, a melanocyte can be a mammalian melanocyte. In someembodiments, an epidermal layer can further comprise a keratinocyte. Insome embodiments, a keratinocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a keratinocyte can expressKRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or acombination thereof. In some embodiments, a keratinocyte can be amammalian keratinocyte. In some embodiments, a mammal can be a non-humanmammal. In some embodiments, a mammal can be a human mammal. In someembodiments, a non-human mammal can be one of a primate, bovine, ovine,porcine, equinine, canine, feline, rodent, or lagomorph. In someembodiments, a fibroblast can be a non-mammalian fibroblast. In someembodiments, a non-mammal can be a fish, a bird or a reptile. In someembodiments, a melanocyte can express Sox-10, MITF-M, gp-100, DCT, TYR,MLANA or a combination thereof. In some embodiments, an epidermal layercan comprise a hair follicle. At least about 2% of cells comprised in anartificial epidermal layer can be differentiated from an inducedpluripotent stem cell. At least about 10% of cells comprised in anartificial epidermal layer can be differentiated from an inducedpluripotent stem cell. At least about 50% of cells comprised in aartificial epidermal layer can be differentiated from an inducedpluripotent stem cell.

Disclosed herein are layered structures. In some embodiments, a layeredstructure can comprise an artificial epidermal layer. In someembodiments, an epidermal layer can comprise a hair follicle cell. Insome embodiments, a layered structure can comprise an artificial dermallayer. In some embodiments, a dermal layer can comprise a fibroblast. Insome embodiments, a fibroblast can be differentiated from an inducedpluripotent stem cell. In some embodiments, a fibroblast can expressCD10, CD73, CD44, CD90, type I collagen, type III collagen,prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, an epidermal layer can further comprise a keratinocyte. Insome embodiments, a keratinocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a keratinocyte can expressKRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or acombination thereof. In some embodiments, a hair follicle cell can be adermal papilla cell, outer root sheath cell or a combination thereof. Insome embodiments, an epidermal layer can further comprise a melanocyte.In some embodiments, a melanocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a melanocyte can expressSox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. Insome embodiments, a layered structure can be pigmented. In someembodiments, an epidermal layer can be stratified. In some embodiments,a layered structure can comprise a basement membrane substitute. In someembodiments, a basement membrane substitute can be between an epidermallayer and a dermal layer. In some embodiments, a basement membranesubstitute can comprise a dried acellular amniotic membrane. In someembodiments, a layered structure can further comprise a scaffold. Insome embodiments, a scaffold can be natural or synthetic. In someembodiments, a scaffold can comprise silk. In some embodiments, ascaffold can comprise chitosan. In some embodiments, a dermal layer canbe upon a scaffold. In some embodiments, a layered structure cancomprise one or more components selected from a group consisting ofkeratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan,glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fattyacid, carbohydrate, or a combination thereof. In some embodiments, alayered structure can further comprise two or more dermal layers. Insome embodiments, a layered structure can further comprise a hairfollicle. In some embodiments, a layered structure can further comprisea fur. At least about 2% of cells comprised in a layered structure canbe differentiated from an induced pluripotent stem cell. At least about10% of cells comprised in a layered structure can be differentiated froman induced pluripotent stem cell. At least about 50% of cells comprisedin layered structure can be differentiated from an induced pluripotentstem cell.

Disclosed herein are methods for making a layered structure. In someembodiments, the method can comprise placing an artificial epidermallayer comprising a hair follicle cell upon an artificial dermal layercomprising a cell differentiated from an induced pluripotent stem cellthereby forming a layered structure. In some embodiments, a celldifferentiated from an induced pluripotent stem cell can be afibroblast, melanocyte, keratinocyte or a combination thereof. In someembodiments, a cell differentiated from an induced pluripotent stem cellcan be a fibroblasts. In some embodiments, a fibroblast can expressCD10, CD73, CD44, CD90, type I collagen, type III collagen,prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, an epidermal layer can further comprise a keratinocyte. Insome embodiments, a keratinocyte can be differentiated from an inducedpluripotent stem cell. In some embodiments, a keratinocyte can expressKRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or acombination thereof. In some embodiments, a hair follicle cell cancomprise a dermal papilla cell, an outer root sheath cell or acombination thereof. In some embodiments, an epidermal layer can furthercomprise a melanocyte. In some embodiments, a melanocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a melanocyte can express Sox-10, MITF-M, gp-100, DCT, TYR,MLANA or a combination thereof. In some embodiments, a dermal layer canbe cultured with a supplement. In some embodiments, a supplement cancomprise collagen, fibrin, growth factors, ascorbic acid, dextransulphate, carrageenan or a combination thereof. In some embodiments, asupplement can be a natural supplement. In some embodiments, asupplement can be a synthetic supplement. In some embodiments, a dermallayer can be cultured upon a scaffold. In some embodiments, a scaffoldcan be natural or synthetic. In some embodiments, a scaffold cancomprise silk. In some embodiments, a scaffold can comprise chitosan. Insome embodiments, a dermal layer can be placed upon a scaffold. In someembodiments, an epidermal layer can be stratified. In some embodiments,a dermal layer can be cultured upon a second dermal layer. In someembodiments, a dermal layer can be cultured in vivo. In someembodiments, a dermal layer may not be cultured upon a collagen matrix.In some embodiments, a thickness of a dermal layer can range from about0.02 mm to about 5 mm. In some embodiments, a thickness of a dermallayer can range from about 0.1 mm to about 0.5 mm. In some embodiments,a thickness of an epidermal layer can range from about 0.01 mm to about2 mm. In some embodiments, a thickness of an epidermal layer can rangefrom about 0.1 mm to about 0.2 mm. At least about 2% of cells comprisedin a layered structure can be differentiated from an induced pluripotentstem cell. At least about 10% of cells comprised in a layered structurecan be differentiated from an induced pluripotent stem cell. At leastabout 50% of cells comprised in layered structure can be differentiatedfrom an induced pluripotent stem cell.

Disclosed herein are layered structures. In some embodiments, a layeredstructure can comprise an artificial epidermal layer comprising a hairfollicle cell and a keratinocyte or a melanocyte; an artificial dermallayer comprising a fibroblast, wherein a fibroblast, a keratinocyte or amelanocyte can be differentiated from an induced pluripotent stem cell,wherein a melanocyte expresses Sox-10, MITF-M, gp-100, DCT, TYR, MLANAor a combination thereof, wherein a fibroblast expresses CD10, CD73,CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylasebeta, or a combination thereof, wherein a keratinocyte expresses KRT14,p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combinationthereof. In some embodiments, a layered structure can comprise anartificial epidermal layer. In some embodiments, an epidermal layer cancomprise a hair follicle cell. In some embodiments, an epidermal layercan comprise a keratinocyte or a melanocyte. In some embodiments, alayered structure can comprise an artificial dermal layer. In someembodiment, a dermal layer can comprise a fibroblast. In someembodiments, a fibroblast, a keratinocyte or a melanocyte can bedifferentiated from an induced pluripotent stem cell. In someembodiments, a melanocyte can express Sox-10, MITF-M, gp-100, DCT, TYR,MLANA or a combination thereof. In some embodiments, a fibroblast canexpress CD10, CD73, CD44, CD90, type I collagen, type III collagen,prolyl-4-hydroxylase beta, or a combination thereof. In someembodiments, a keratinocyte can express KRT14, p63, DSG3, ITGB4, LAMA5,KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments,a thickness of a dermal layer can range from about 0.02 mm to about 5mm. In some embodiments, a thickness of a dermal layer can range fromabout 0.1 mm to about 0.5 mm. In some embodiments, a thickness of anepidermal layer can range from about 0.01 mm to about 2 mm. In someembodiments, a thickness of an epidermal layer can range from about 0.1mm to about 0.2 mm. At least about 2% of cells comprised in a layeredstructure can be differentiated from an induced pluripotent stem cell.At least about 10% of cells comprised in a layered structure can bedifferentiated from an induced pluripotent stem cell. At least about 50%of cells comprised in layered structure can be differentiated from aninduced pluripotent stem cell.

Disclosed herein are artificial epidermal layer. An artificial epidermallayer can comprise a stratum corneum. An artificial epidermal layer cancomprise a stratum granulosum. An artificial epidermal layer cancomprise a stratum spinosum. An artificial epidermal layer can comprisea stratum basale. In some embodiments a stratum corneum, a stratumgranulosum, a stratum spinosum, or a stratum basale can be organized asdepicted in FIG. 6A, or FIG. 8A. A thickness of a stratum corneum canrange from about 0.01 mm to about 0.05 mm. A thickness of astratumgranulosum can range from about 0.01 mm to about 0.15 mm. A thickness ofa stratum spinosum can range from about 0.01 mm to about 0.15 mm. Athickness of said stratum basale can range from about 0.01 mm to about0.15 mm. A thickness of a stratum corneum can range from about 4% toabout 20% of an artificial epidermal layer. A thickness of a stratumgranulosum can range from about 4% to about 60% of a artificialepidermal layer. A thickness of a stratum spinosum can range from about4% to about 40% of a artificial epidermal layer. A thickness of astratum basale can range from about 4% to about 40% of an artificialepidermal layer. At least about 2% of cells comprised in an artificialepidermal layer can be differentiated from an induced pluripotent stemcell. At least about 10% of cells comprised in an artificial epidermallayer can be differentiated from an induced pluripotent stem cell. Atleast about 50% of cells comprised in a artificial epidermal layer canbe differentiated from an induced pluripotent stem cell.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features described herein are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the features described herein will be obtained byreference to the following detailed description that sets forthillustrative examples, in which the principles of the features describedherein are utilized, and the accompanying drawings of which:

FIG. 1 illustrates a synthetic leather production schematic.

FIGS. 2A-2F illustrate a layered structure. FIG. 2A depicts a layeredstructure comprising an epidermal layer and a dermal layer on ascaffold. FIG. 2B depicts a layered structure comprising an epidermallayer, a basement membrane substitute and a dermal layer on a scaffold.FIG. 2C depicts a layered structure comprising an epidermal layer andmultiple dermal layers on a scaffold. FIG. 2D depicts a layeredstructure comprising an epidermal layer, a basement membrane substituteand multiple dermal layers on a scaffold. FIG. 2E depicts a layeredstructure comprising an epidermal layer, a basement membrane substituteand multiple dermal layers. FIG. 2F depicts a layered structurecomprising an epidermal layer and multiple dermal layers.

FIG. 3 illustrates a layered structure development.

FIGS. 4A-4C illustrate a comparative analysis of leather (FIG. 4A),native skin (FIG. 4B) and epidermal equivalent (FIG. 4C).

FIGS. 5A-5C illustrate a comparative analysis of stratum corneum ofnative skin and epidermal equivalent. FIG. 5A depicts an epidermalsurface image. FIG. 5B depicts a corneo-desmosome image. FIG. 5C depictsa CDSN/Hoechst image.

FIGS. 6A-6E illustrate a comparative analysis of stratum granulosum ofnative skin and epidermal equivalent. FIG. 6A depicts a Loricrin (LOR)staining. FIG. 6B depicts an epidermal Ca⁺⁺ gradient captured ontransmission electron microscopy as electron-dense precipitates. FIG. 6Cdepicts an assessment of permeability barrier integrity by lanthanumperfusion. FIG. 6D illustrates that tight junction protein 1/zonulaoccludens-1 (TJP1/ZO-1) anchors tight junction strand proteins, whichcan be fibril-like structures within the lipid bilayer, to the actincytoskeleton. FIG. 6E illustrates that Filaggrin (FLG) monomers,tandemly clustered into a large, 350 kDa protein precursor known asprofilaggrin, are present in the keratohyalin granules in cells of theSG.

FIGS. 7A-7C illustrate a Lipid bilayer formation in native skin andepidermal equivalent assessed with TEM. FIG. 7A depicts normal lipidsecretion at the border of SC and SG. FIG. 7B depicts lamellar bodies inthe SG. FIG. 7C depicts normal lipid bilayer (LB) morphology of nativeskin.

FIGS. 8A-8C illustrate a comparative analysis of markers of suprabasallayers of native skin and epidermal equivalent, including Keratin 10(KRT10; FIG. 8A), keratin 1 (KRT1; FIG. 8B), desmocollin 1 (DCL1; FIG.8C), markers of suprabasal layers. FIG. 8D depicts Desmosomes in bothnative skin in vivo and epidermal equivalents generated in vitro.

FIGS. 9A-9C illustrate a comparative analysis of stratum basale ofnative skin and epidermal equivalent. MKI67 (FIG. 9A), a marker ofproliferation, keratin 14 (KRT14; FIG. 9B), and transcription factorTP63 (FIG. 9C) show typical basal layer distribution in both native skinin vivo (left side of panel) and epidermal equivalents generated invitro. FIG. 9D depicts hemidesmosomes in both native skin in vivo andepidermal equivalents generated in vitro.

FIGS. 10A-10F illustrate a comparative analysis of extracellular matrixcomponents of basement membrane. FIG. 10A depicts Integrin β1expression. FIG. 10B depicts fibronectin expression.

FIG. 10C depicts collagen IV expression. FIG. 10D depicts collagen VIexpression. FIG. 10E depicts collagen VII expression. FIG. 10F depictsLaminin 5 expression.

FIGS. 11A-11I illustrate a structural analysis of full-thickness skinequivalent (FSE). FIGS. 11A and 11B depict cross sections of FSEdisplays distinct cellular layers of epidermis under 2600× magnification(FIG. 11A) and 5200× magnification (FIG. 11B). FIG. 11C depicts asurface of an FSE at 900× magnification. FIGS. 11D-1F depictlongitudinal sections of dermal scaffold with residing dermalfibroblasts and rich extracellular matrix at 91× magnification (FIG.11D), 162× magnification (FIG. 11E) and 405× magnification (FIG. 11F).FIGS. 11G-11I depict dermal scaffolds with residing dermal fibroblastsand rich extracellular matrix at 80× magnification (FIG. 11G), 695×magnification (FIG. 11H) and 2700× magnification (FIG. 11I).

FIGS. 12A-12R illustrate a time-course of engineering dermal equivalent.FIGS. 12A-12I depict day 2 after seeding dermal fibroblasts ontoscaffold at 36× magnification (FIG. 12A), 695× magnification (FIG. 12B),1470× magnification (FIG. 12C), 7750× magnification (FIG. 12D), 2320×magnification (FIG. 12E), 2420× magnification (FIG. 12F), 6560×magnification (FIG. 12G), 17000× magnification (FIG. 12H) and 22000×magnification (FIG. 12I). FIGS. 12J-12 R depict day 7 after seedingdermal fibroblasts onto scaffold at 64× magnification (FIG. 12J), 100×magnification (FIG. 12K), 364× magnification (FIG. 12L), 82×magnification (FIG. 12M), 253× magnification (FIG. 12N), 3940×magnification (FIG. 12O), 5550× magnification (FIG. 12P), 9440×magnification (FIG. 12Q) and 21680 magnification (FIG. 12R).

DETAILED DESCRIPTION OF THE DISCLOSURE

Several aspects are described below with reference to exampleapplications for illustration. It should be understood that numerousspecific details, relationships, and methods are set forth to provide afull understanding of the features described herein. One having ordinaryskill in the relevant art, however, will readily recognize that thefeatures described herein can be practiced without one or more of thespecific details or with other methods. The features described hereinare not limited by the illustrated ordering of acts or events, as someacts can occur in different orders and/or concurrently with other actsor events, unless otherwise specifically indicated. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the features described herein.

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising”.

In this disclosure the term “about” or “approximately” can mean a rangeof up to 10% of a given value. In this disclosure the term“substantially” refers to something that can be done to a great extentor degree.

As used herein, the term “pluripotent stem cell” can refer to anyprecursor cell that has the ability to form any adult cell.

As used herein, the term “embryonic stem cells” or “ES cells” or “ESC”can refer to precursor cells that have the ability to form any adultcell.

As used herein, the term “induced pluripotent stem cells” or “iPS cells”or “iPSCs” can refer to a type of pluripotent stem cell artificiallyderived from a non-pluripotent cell (e.g., an adult somatic cell).Induced pluripotent stem cells can be identical to embryonic stem cellsin the ability to form any adult cell, but are not derived from anembryo. In some cases, IPSC cells disclosed herein can be IPSC cells.

As used herein, the term “synthetic leather” can mean that the skinequivalents described herein can serve as a skin equivalent for anymammal or non-mammal. Embodiments can be practiced with human andnon-human mammals, such as non-human primates and members of the bovine,ovine, porcine, equinine, canine and feline species as well as rodentssuch as mice, rats and guinea pigs, members of the lagomorph familyincluding rabbit; and non-mammals such as fish including shark andstingray, birds including ostrich and reptiles including lizards, snakesand crocodiles. The particular mammalian synthetic leather which will beformed can be dependent on the source of the cells used in embodimentsdescribed herein, e.g. Keratinocytes and fibroblasts, e.g., when bovinekeratinocytes and fibroblasts are used to form a skin equivalent, abovine synthetic leather can be formed.

Overview

Disclosed herein are synthetic leathers, artificial epidermal layers,artificial dermal layers, layered structures, products made thereof andmethods of producing the same. In certain cases, disclosed herein aresynthetic leathers. In some cases, a synthetic leather comprises one ora plurality of layers. In some cases, one or a plurality of layerscomprises cells, wherein said cells are cultured in vitro. In somecases, the methods described herein provide high-throughput methods thatreliably, accurately, and reproducibly scale up to commercial levels theproduction of synthetic leather. Advantages of the synthetic leather,engineered epidermal equivalent, engineered full thickness skinequivalent and methods of making the same disclosed herein include, butare not limited to, production of customized tissues in a reproducible,high throughput and easily scalable fashion with appealing appearance,texture, thickness, and durability. As used herein, full thickness skinequivalent can comprise at least one dermal layer and at least oneepidermal layer. As used herein, full thickness skin equivalent and fullskin equivalent can be used interchangeably.

A synthetic leather disclosed herein can comprise a layer of artificialdermal layer comprising a fibroblast and an artificial epidermal layercomprising a keratinocyte. The dermal layer and the epidermal layer canform a layered structure. A synthetic leather can comprise one or morelayered structure. The synthetic leather can be tanned and furtherprocessed. The cells forming the synthetic layer can be differentiatedfrom stem cells, e.g., induced pluripotent stem cells (iPSC). The dermallayer can be placed on a scaffold, such as silk, to achieve naturalleather thickness and texture.

Also disclosed herein are methods of making a synthetic leather. Themethod can comprise forming a layered structure comprising an artificialdermal layer and an artificial epidermal layer, and tanning the layeredstructure. The methods can also comprise further processing theartificial dermal layers and epidermal layers, e.g., to achieve naturalleather thickness and texture.

Synthetic Leather

A synthetic leather can comprise one or more cell layers. For example, asynthetic leather can comprise one or more of: a dermal layer, anepidermal layer, and a basement membrane or a basement membranesubstitute. A synthetic leather can further comprise hypodermis, scale,scute, osteoderm, or a combination thereof. In some cases, a syntheticlayer comprises a full thickness skin equivalent. Such full thicknessskin equivalent can comprise any one or combination of the layersdisclosed herein. A portion of one or more cell layers in a syntheticleather can be removed, e.g., by shaving. In some cases, a syntheticleather can be tanned. The tanning can be performed after formation ofone or more of the cell layers or layered structures. The tanning can beperformed after at least a portion of a cell layer can be removed from asynthetic leather. In some cases, a synthetic leather can be furtherprocessed. In some cases, a synthetic leather can comprise a hairfollicle cell and a melanocyte. The hair follicle cell and/or themelanocyte can be differentiated from a stem cell (e.g., an iPSC).

In some embodiments, a tanned synthetic leather can comprise a layeredstructure. A layered structure can comprise an artificial dermal layercomprising a fibroblast. A layered structure can also comprise anartificial epidermal layer comprising a keratinocyte. In some cases, alayered structure can comprise an artificial dermal layer comprising afibroblast and an artificial epidermal layer comprising a keratinocyte.In some cases, a fibroblast or a keratinocyte can be differentiated froman induced pluripotent stem cell.

In some cases, a tanned synthetic leather can comprise at least part ofa dermal layer. In some cases, a tanned synthetic leather does notcomprise a dermal layer. In some cases, a dermal layer can be removed.

Dermal Layer

A synthetic leather can comprise a dermal layer (e.g., an artificialdermal layer). A dermal layer can be an engineered dermis equivalent,e.g., an artificial dermal layer formed in vitro.

A dermal layer can comprise cells of connective tissue. For example, adermal layer can comprise fibroblasts. Fibroblasts in the dermal layercan express one or more markers including, but not limited to, clusterof differentiation 10 (CD10), cluster of differentiation 73 (CD73),cluster of differentiation 44 (CD44), cluster of differentiation 90(CD90), type I collagen, type III collagen, and prolyl-4-hydroxylasebeta fibroblasts. In some cases, a dermal layer also comprises othertypes of cells, such as immune cells, macrophages, adipocytes, or acombination thereof.

A dermal layer can further comprise matrix components in addition tocells. Examples of matrix components include but are not limited to anyone or more of collagen, elastin, and extrafibrillar matrix, anextracellular gel-like substance primarily composed ofglycosaminoglycans (e.g., hyaluronan), proteoglycans, and glycoproteins.

A dermal layer can comprise a matrix support. A matrix support can be ascaffold. The matrix support can comprise contracted collagen gels.Alternatives to a pure collagen matrix can be a polyglygolic acid mesh,e.g., as described in Hansbrough, et al., J. Burn Care Rehabil.,15:346-53 (1994), or collagen and glycosaminoglycan matrix covered witha silastic membrane (C-GAG), e.g., as described in Burke, et al., Ann.Surg., 194:413-420 (1981) or various biopolymers, e.g. chitosan asdescribed in Kellouche, et al., Biochem Biophys Res Commun., 363:472-478(2007). In some cases, the matrix can be seeded with fibroblasts, e.g.,to give rise to organotypic models. Naturally derived dermis, fromallogenic cadaver skin can also be used with keratinocyte sheets. Avariation of this technique can use lyophilized devitalized dermis fromcadaver skin to support the keratinocyte sheets.

The thickness of leather units may be reported in millimeters, ounces,or irons. (One ounce equals 1/64 in. or 0.0156 in. or 0.396 mm. One ironequals 1/48 in. or 0.0208 in. or 0.53 mm.)

The thickness of a dermal layer can be engineered to fit the function oruse of a synthetic leather. A dermal layer can have a thickness fromabout 0.01 mm to about 50 mm. For example, a dermal layer can have athickness from about 0.01 mm to about 10 mm, from about 0.01 mm to about8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, fromabout 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8mm, or from about 0.1 to about 0.5 mm. For example, a dermal layer canhave a thickness from about 0.02 mm to 5 mm. For example, a dermal layercan have a thickness from about 0.1 mm to 0.5 mm. For example, a dermallayer can have a thickness from about 0.2 mm to 0.5 mm. In some cases,the thickness of a dermal layer can be at least 0.001 mm, 0.01 mm, 0.02mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm,8 mm, or 10 mm. In some cases, the thickness of a dermal layer can be atmost 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm,0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In someembodiments, a dermal layer can have a thickness of at least about 50mm.

The length of a dermal layer can be engineered to fit the function oruse of a synthetic leather. A dermal layer can have a length from about0.01 mm to about 50 m. For example, a dermal layer can have a lengthfrom about 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm,from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 toabout 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8mm, or from about 0.1 to about 0.5 mm. For example, a dermal layer canhave a length from about 0.02 mm to 5 mm. For example, a dermal layercan have a length from about 0.1 mm to 0.5 mm. For example, a dermallayer can have a length from about 0.2 mm to 0.5 mm. In some cases, thelength of a dermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm,0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8mm, or 10 mm. In some cases, the length of a dermal layer can be at most50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments,a dermal layer can have a length of at least about 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, a dermallayer can have a length of at least about 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700 cm. In some embodiments, a dermal layer can havea length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

The width of a dermal layer can be engineered to fit the function or useof a synthetic leather. A dermal layer can have a width from about 0.01mm to about 50 m. For example, a dermal layer can have a width fromabout 0.01 mm to about 10 mm, from about 0.01 mm to about 8 mm, fromabout 0.01 to about 5 mm, from about 0.02 to about 5 mm, from about 0.05to about 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8 mm, orfrom about 0.1 to about 0.5 mm. For example, a dermal layer can have awidth from about 0.02 mm to 5 mm. For example, a dermal layer can have awidth from about 0.1 mm to 0.5 mm. For example, a dermal layer can havea width from about 0.2 mm to 0.5 mm. In some cases, the width of adermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. Insome cases, the width of a dermal layer can be at most 50 mm, 40 mm, 20mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, a dermal layercan have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 700, 1000 mm. In some embodiments, a dermal layer can have awidth of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700 cm. In some embodiments, a dermal layer can have a width of atleast about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

A synthetic leather can comprise one or more dermal layers. For example,a synthetic leather can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 40, 60, 80, or 100 dermal layers. When a syntheticleather comprises more than one dermal layer, a dermal layer can beplaced upon another dermal layer. For example, a synthetic leather cancomprise two dermal layers, e.g., a first dermal layer and a seconddermal layer. The first dermal layer can be placed upon the seconddermal layer.

A dermal layer can be stratified, e.g., having a plurality of sublayers.The sublayers can have different compositions, e.g., differentconcentrations of the fibers. The sublayers of a dermal layer can havedifferent thicknesses and densities. For example, a dermal layer canhave a papillary dermal layer, a reticular dermal layer, or anycombination thereof. A papillary dermal layer can comprise loose areolarconnective tissue and/or loosely arranged fibers, e.g., collagen fibers.A reticular dermal layer can comprise dense irregular connective tissue,including collagen fibers and dermal elastic fibers.

A dermal layer can comprise a free collagen matrix or lattice, which canbe contractile in all directions, and homogeneous. Fibroblasts, andwhere appropriate other types of cells of the dermis, can be distributedin a continuous collagen gel. The dermis equivalent can comprise atleast one matrix of collagen type I in which the fibroblasts aredistributed. It can also contain other extracellular matrixconstituents. Extracellular matrix constituent can include collagens,e.g., collagen IV, laminins, entactin, fibronectin, proteoglycans,glycosaminoglycans or hyaluronic acid. A dermal layer can containcollagen type IV and laminin, entactin, or a combination thereof. Theconcentrations of these various constituents can be adjusted. Forexample, the concentration of laminin can be from about 1% to about 15%of the final volume. For example, the concentration of collagen IV canbe from about 0.3% to about 4.5% of the final volume. For example, theconcentration of entactin can be from about 0.05% to about 1% of thefinal volume. The collagen used can be collagen of bovine origin, fromrat tail or from fish, or any other source of natural collagen orcollagen produced by genetic engineering which allows contraction in thepresence of fibroblasts. In some embodiments, collagen can be from anunnatural source. The matrix can be a gel of collagen which may nottaut, obtained by contraction both horizontally and vertically, whichdoes not impose a preferential organization of the fibroblasts. Such amatrix, also termed “free”, may not adhere to the support and thevolumes thereof can be modified without limit, conferring on it avarying thickness and diameter. The thickness of the dermis equivalentcan be at least 0.05 cm and in some cases approximately from 0.05 to 2cm. The thickness can also be increased without harming the advantageousproperties of the skin equivalent or synthetic leather. In some cases,the thickness can be from about 3 mm to about 20 cm or more.

Epidermal Layer

A synthetic leather can comprise an epidermal layer (e.g., an artificialepidermal layer). An epidermal layer can be an engineered epidermisequivalent, e.g., an artificial epidermal layer formed in vitro.

An epidermal layer can comprise one or more types of cells, includingkeratinocytes, melanocytes, Langerhans cells, Merkel cells, andinflammatory cells. For example, an epidermal layer can comprisekeratinocytes. Keratinocytes in an epidermal layer can includeepithelial keratinocytes, basal keratinocytes, proliferating basalkeratinocytes, differentiated suprabasal keratinocytes, or anycombination thereof.

In some cases, an epidermal layer comprises at least basalkeratinocytes, e.g., keratinocytes which are not differentiated. Anepidermal layer can further comprise partially differentiatedkeratinocytes as well as fully differentiated keratinocytes. In one ormore epidermal layers in a synthetic leather there can be a transitionfrom undifferentiated basal keratinocytes to fully differentiatedkeratinocytes as one progresses from the dermal-epidermal junction wherethe basal keratinocytes are localized.

Basal keratinocytes can express hemidesmosomes, which serve to helpsecure the epidermal and dermal layers together. Basal keratinocytes canalso serve to regenerate the skin. An epidermal layer in a syntheticleather herein can have basal keratinocytes that serve these functions.Thus, a synthetic leather comprising such basal keratinocytes can becapable of regeneration. Other distinctions between basal keratinocytesand differentiated keratinocytes in one or more epidermal layers in asynthetic leather can be that both E- and P-cadherin's are present inepidermal keratinocytes along the basal membrane zone (BMZ), butkeratinocytes which are differentiated and located away from the BMZonly express E-cadherin.

The basal keratinocytes of an epidermal layer can be aligned in a layerin direct contact with the dermal layer, serving as the boundary betweenthe differentiated keratinocytes and the fibroblasts. In alternativecases, there are gaps between the basal keratinocytes and the dermallayer. Still further, there can be gaps between the basal keratinocytesand other basal keratinocytes, leaving gaps between the differentiatedkeratinocytes and the dermal layer. In these latter cases where thereare gaps between the basal or differentiated keratinocytes and thedermal layer, the dermal and epidermal layers are not uniformly incontact with one another, but are adjacent to each other. They areadjacent in that there can be generally fluid, but substantially noother intervening materials such as layers of cells, collagen, matricesor other supports between the dermal and epidermal layers.

Keratinocytes in an epidermal layer can express one or more markers.Such markers include, but are not limited to, Keratin 14 (KRT14), tumorprotein p63 (p63), Desmoglein 3 (DSG3), Integrin, beta 4 (ITGB4),Laminin, alpha 5 (LAMA5), Keratin 5 (KRT5), an isoform of tumor proteinp63 (e.g., TAp63), Laminin, beta 3 (LAMB3), and Keratin 18 (KRT18).

The thickness of an epidermal layer can be engineered to fit thefunction or use of the synthetic leather. An epidermal layer can have athickness from about 0.001 mm to about 10 mm. For example, an epidermallayer can have a thickness from about 0.005 mm to about 10 mm, fromabout 0.005 mm to about 5 mm, from about 0.005 mm to about 2 mm, fromabout 0.01 mm to about 10 mm, from about 0.01 mm to about 5 mm, fromabout 0.01 mm to about 2 mm, from about 0.01 mm to about 1, from about0.01 mm to about 0.8 mm, from about 0.01 mm to about 0.4 mm, from about0.01 mm to about 0.2 mm, from about 0.01 mm to about 0.1 mm, from about0.05 mm to about 0.4 mm, from about 0.05 mm to about 0.2 mm, from about0.05 mm to about 0.1 mm, from about 0.1 mm to about 0.4 mm, from about0.1 mm to about 0.2 mm, from about 0.08 mm to about 1 mm, or from about0.05 mm to about 1.5 mm. For example, an epidermal layer can have athickness from about 0.01 mm to about 2 mm. For example, an epidermallayer can have a thickness from about 0.1 mm to about 0.22 mm. In somecases, the thickness of an epidermal layer can be at least 0.001 mm,0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the thickness of thedermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or0.01 mm. In some cases, thickness values described herein can be thethickness of an epidermal layer and a basement membrane substitute.

The length of an epidermal layer can be engineered to fit the functionor use of a synthetic leather. An epidermal layer can have a length fromabout 0.01 mm to about 50 m. For example, an epidermal layer can have alength from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, fromabout 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8mm, or from about 0.1 to about 0.5 mm. For example, an epidermal layercan have a length from about 0.02 mm to 5 mm. For example, an epidermallayer can have a length from about 0.1 mm to 0.5 mm. For example, anepidermal layer can have a length from about 0.2 mm to 0.5 mm. In somecases, the length of an epidermal layer can be at least 0.001 mm, 0.01mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of an epidermallayer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm,0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm.In some embodiments, an epidermal layer can have a length of at leastabout 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In someembodiments, an epidermal layer can have a length of at least about 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In someembodiments, an epidermal layer can have a length of at least about 50,60, 70, 80, 90, 100, 200, 300, 400 m.

The width of an epidermal layer can be engineered to fit the function oruse of a synthetic leather. An epidermal layer can have a width fromabout 0.01 mm to about 50 m. For example, an epidermal layer can have awidth from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, fromabout 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8mm, or from about 0.1 to about 0.5 mm. For example, an epidermal layercan have a width from about 0.02 mm to 5 mm. For example, an epidermallayer can have a width from about 0.1 mm to 0.5 mm. For example, anepidermal layer can have a width from about 0.2 mm to 0.5 mm. In somecases, the width of an epidermal layer can be at least 0.001 mm, 0.01mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of an epidermal layercan be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. Insome embodiments, an epidermal layer can have a width of at least about50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In someembodiments, an epidermal layer can have a width of at least about 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In someembodiments, an epidermal layer can have a width of at least about 50,60, 70, 80, 90, 100, 200, 300, 400 m.

A synthetic leather can comprise one or more epidermal layers. Forexample, a synthetic leather can have at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 40, 60, 80, or 100 epidermal layers.When a synthetic leather comprises more than one epidermal layer, oneepidermal layer can be placed upon another epidermal layer. For example,a synthetic leather can comprise two epidermal layers, e.g., a firstepidermal layer and a second epidermal layer. The first epidermal layercan be placed upon the second epidermal layer.

An epidermal layer can be stratified, e.g., having a plurality ofsublayers. The sublayers can have different cell compositions, e.g.,different types of keratinocytes. The sublayers of an epidermal layercan have different thicknesses and/or densities. For example, anepidermal layer can have one or more of cornified layer (stratumcorneum), clear/translucent layer (stratum lucidum), granular layer(stratum granulosum), spinous layer (stratum spinosum), basal/germinallayer (stratum basale/germinativum), or any combination thereof. In somecases, an epidermal layer comprises functional epidermal permeabilitybarrier (e.g., organized lipid bilayers in stratum corneum). In somecases, a stratum corneum, stratum lucidum, stratum granulosum, stratumspinosum, or stratum basale/germinativum, can have a thickness of about0.0001 mm to about 5 mm. In some cases, a stratum corneum, stratumlucidum, stratum granulosum, stratum spinosum, or stratumbasale/germinativum, can have a thickness of at least about 0.001 mm,0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.15 mm, 0.2 mm, 0.4 mm, 0.8mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, a stratum corneum,stratum lucidum, stratum granulosum, stratum spinosum, or stratumbasale/germinativum, can have a thickness of at most about 50 mm, 40 mm,20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.15 mm,0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm.

An epidermal layer can further comprise cells producing pigments, e.g.,melanin. Such pigment-producing cells can be melanocytes. Melanocytes inthe epidermal layer can express one or more markers. Such markers caninclude, but are not limited to, SRY-box containing gene 10 (Sox-10),Microphthalmia-associated transcription factor (MITF-M), premelanosomeprotein (gp-100), Dopachrome tautomerase (DCT), Tyrosinase (TYR), andMelan-A (MLANA).

Cells in Synthetic Leather

A synthetic leather can comprise cells in the dermal layer and epidermallayer disclosed herein. In some cases, a synthetic leather alsocomprises hair follicle cells, endothelial cells, dermal papilla cells,immune system cells (such as lymphocytes, dendritic cells, macrophagesor Langerhans cells), adipocytes, nerve cells, and a mixture thereof.

One or more cells in a synthetic leather can be genetically engineeredcells. The term “genetically engineered” can refer to a man-madealteration to the nucleic acid content of a cell. Therefore, geneticallyengineered cells can include cells containing an insertion, deletion,and/or substitution of one or more nucleotides in the genome of a cellas well as alterations including the introduction of self-replicatingextrachromosomal nucleic acids inserted into the cell. Geneticallyengineered cells also include those in which transcription of one ormore genes has been altered, e.g., increased or reduced.

In some cases, a synthetic leather has at least one of the components ofnative skin such as melanocytes, hair follicles, sweat glands and nerveendings. In certain cases, a synthetic leather can be distinguished fromnormal native skin by its lack of at least one of these components. Insome cases displaying abnormal phenotypes or having at least one cellwith an altered genotype, a synthetic leather can include all of thesecomponents.

In some case, additional components can be added to a synthetic leather.Such additional components can include myoepithelial cells, duct cells,secretory cells, alveolar cells, langerhans cells, Merkel cells,adhesions, mammary glands, or any mixture thereof. In some cases, asynthetic leather comprises one or more of: neural cells, connectivetissue (including bone, cartilage, cells differentiating into boneforming cells and chondrocytes, and lymph tissues), epithelial cells(including endothelial cells that form linings in cavities and vesselsor channels, exocrine secretory epithelial cells, epithelial absorptivecells, keratinizing epithelial cells, and extracellular matrix secretioncells), and undifferentiated cells (such as embryonic cells, stem cells,and other precursor cells).

A synthetic leather can comprise hair follicles. A hair follicle cancomprise one or more structures, including papilla, matrix, root sheath,bulge, infundibulum, the arrector pili muscles, the sebaceous glands,and the apocrine sweat glands. A hair follicle can comprise one or morehair follicle cells, including dermal papilla cell, outer root sheathcell, or any combination thereof. In some cases, a hair follicle can bein an epidermal layers. In some cases, a hair follicle can be in adermal layer. A hair follicles cell can be differentiated from aprogenitor, e.g., a stem cell such as an iPSC. In some embodiments, atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% of hair follicle cells can be differentiatedfrom induced pluripotent stem cells.

In some embodiments, a synthetic leather can be devoid of hair, bloodvessels, sebaceous glands, hair follicle, oil glands, nerve, or acombination thereof.

In some cases, a synthetic leather can comprise hairs, e.g., in one ormore layered structures. For example, a synthetic leather can comprisefur. The hairs (e.g., fur) can be natural, synthetic, or a combinationthereof. The hairs (e.g., fur) can be grown from cells in the syntheticleather, or added to synthetic leather from an exogenous source. Inother cases, a synthetic leather may not have any hairs.

Stem Cells

One or more cells in a synthetic leather can be differentiated fromprogenitor cells, such as stem cells. For example, fibroblasts in asynthetic leather can be differentiated from stem cells. For example,keratinocytes in a synthetic leather can be differentiated from stemcells. For example, melanocytes in a synthetic leather can bedifferentiated from stem cells. In some embodiments, at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100% of cells disclosed herein can be differentiated from stemcells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of fibroblastscan be differentiated from induced pluripotent stem cells. In someembodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of keratinocytes can bedifferentiated from induced pluripotent stem cells. In some embodiments,at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100% of melanocytes cells can be differentiatedfrom induced pluripotent stem cells.

Stem cells can be embryonic stem cells (ESCs), adult stem cells (i.e.,somatic stem cells) or induced pluripotent stem cells (iPSCs). In someembodiments, a stem cell can be totipotent, pluripotent or multipotentfor example adult stem cells and cord blood stem cells). Embryonic stemcells can be derived from fertilized embryos that are less than one weekold. Induced pluripotent stem cells can be obtained through the inducedexpression of one or more of Oct3, Oct4, Sox2, Klf4, and c-Myc genes inany somatic cell (e.g., adult somatic cell) such as fibroblast. In somecases, one or more other genes can also be induced for reprograming asomatic cell to an induced pluripotent stem cell. Examples of such genesinclude NANOG, UTF1, LIN28, SALL4, NR5A2, TBX3, ESSRB, DPPA4, SV40LT,REM2, MDM2, and cyclin D1.

Various delivery methods can be used to modulate the expression of genesto reprogram a somatic cell to an iPSC. Exemplary delivery methodsinclude naked DNA delivery, adenovirus, electrical delivery, chemicaldelivery, mechanical delivery, polymer based systems, microinjection,retroviruses (e.g., MMLV-derived retroviruses), and lentiviruses (e.g.,excisable lentiviruses). In some cases, induced pluripotent stem cellscan be obtained according to the protocol as described by Takahashi etal., Cell. 2007 Nov. 30; 131(5):861-72 (2007), or by Yu et al., Science318, 1917-1920 (2007) (2007). In some case, somatic cells (e.g., adultsomatic cells) are transfected with viral vectors, such as retroviralvectors, which comprise Oct3, Oct4, Sox2, Klf4, and c-Myc genes. In somecases, Sendai viruses are used as a delivery system, e.g., Sendaiviruses produced by ID Pharma Co., Ltd., Japan.

Sources of Cells

A synthetic leather can comprise cells derived from animals of one ormore species. For example, the cells in a synthetic leather can bederived from mammals, birds, reptiles, amphibian, fish, invertebrates,or any combination thereof.

A synthetic leather can comprise cells derived from mammals, e.g.,mammalian cells, or non-mammals. A mammal can be a non-human mammal. Anon-human mammal can be antelope, bear, beaver, bison, boar, camel,caribou, cat, cattle, deer, dog, elephant, elk, fox, giraffe, goat,hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen,peccary, pig, rabbit, rhino, seal, sheep, squirrel, tiger, whale, wolf,yak, or zebra. In some cases, a mammal can be primate, bovine, ovine,porcine, equinine, canine, feline, rodent, or lagomorph. A non-mammalcan be a fish, a bird or a reptile. In some cases, a mammal can be ahuman. In some embodiments a human can be a celebrity. As used herein,the term “celebrity” can be defined as a person that has come into thecommunity attention by way of notoriety or general fame of previousactivities. A “celebrity” can be associated with industries includingbut not limited to professional and amateur sports, entertainment,music, motion picture, business, print and electronic media, politics,and the like.

A synthetic leather can comprise cells derived from other species. Insome cases, the cells are derived from birds, such as chicken, duck,emu, goose, grouse, ostrich, pheasant, pigeon, quail, or turkey. In somecases, the cells are derived from reptiles such as turtle, snake,crocodile, or alligator. In some cases, the cells are derived fromamphibians such as frog, toad, salamander, or newt. In some cases, thecells are derived from fish, such as anchovy, bass, catfish, carp, cod,eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel,mahi-mahi, manta ray, marlin, orange roughy, perch, pike, pollock,salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia,trout, tuna, or walleye.

In some cases, all cells in a synthetic leather are derived from thesame species. For example, all cells in a synthetic leather can bebovine cells. In other cases, a synthetic leather comprises cellsderived from multiple species. For example, a synthetic leather cancomprise bovine cells and alligator cells. In some cases, a syntheticleather comprises cells derived from at least 2, 3, 4, 5, 6, 7, 8, or 10species.

Progenitors of the cells in a synthetic leather can also be derived fromthe sources described herein. For example, stem cells (e.g., iPSCs),somatic cells (e.g., to be reprogramed to iPSCs), primary cells used insynthetic cells, dermal layer cells, epidermal layer cells, or any cellsin the synthetic and their progenitors thereof can be derived from thesources described herein.

Any cell can be a live cell or a dead cell. When multiple cells arepresent, a cells may be a live cell, may be a dead cell, or anycombination thereof.

Layered Structure

A synthetic leather can comprise one or more layered structures. Alayered structure can be formed by placing a first type of layer upon asecond type of layer. The first type of layer and the second type oflayer can be the same or different. In some cases, a layered structurecan be formed by placing an epidermal layer upon a dermal layer. Forexample, a layered structure can be formed by placing an epidermal layerupon a dermal layer, with a basement membrane substitute in between.

A layered structure can comprise two or more layers. In some cases, alayered structure comprises at least 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 layers. In some cases, alayered structure comprises at least 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 first type of layers, andat least 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 second type oflayers. For example, a layered structure can comprise at least 2, 3, 4,5 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000dermal layers, and at least 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 500, or 1000 layers of epidermal layers.

A layered structure can comprise one or more types of cells describedherein. For example, a layered structure can comprise cells in a dermallayer, such as fibroblasts, cells in an epidermal layer, such askeratinocytes, or any combination thereof. In some cases, a layeredstructure further comprises cells other than fibroblasts andkeratinocytes. For example, a layered structure can comprisemelanocytes.

A layered structure can have a thickness from about 0.001 mm to about100 mm. For example, a layered structure can have a thickness from about0.005 mm to about 50 mm, from about 0.005 to about 10, from about 0.01mm to about 10 mm, from about 0.02 to about 5 mm, from about 0.05 toabout 5 mm, from about 0.1 to about 5 mm, from about 0.1 to about 2 mm,from about 0.1 to about 1 mm, or from about 0.1 to about 0.5 mm. In somecases, the thickness of a layered structure can be at least 0.001 mm,0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1mm, 2 mm, 4 mm, 8 mm, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. Insome cases, the thickness of a layered structure can be at most 100 mm,50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments,a layered structure can have a thickness of at least about 100, 200,300, 400, 500, 600, 700, 800 mm.

The length of a layered structure can be engineered to fit the functionor use of a synthetic leather. A layered structure can have a lengthfrom about 0.01 mm to about 50 m. For example, a layered structure canhave a length from about 0.01 mm to about 10 mm, from about 0.01 mm toabout 8 mm, from about 0.01 to about 5 mm, from about 0.02 to about 5mm, from about 0.05 to about 5 mm, from about 0.1 to about 5 mm, fromabout 0.1 to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 toabout 0.8 mm, or from about 0.1 to about 0.5 mm. For example, a layeredstructure can have a length from about 0.02 mm to 5 mm. For example, alayered structure can have a length from about 0.1 mm to 0.5 mm. Forexample, a layered structure can have a length from about 0.2 mm to 0.5mm. In some cases, the length of a layered structure can be at least0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm,0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of alayered structure can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm,2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm,or 0.01 mm. In some embodiments, a layered structure can have a lengthof at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000mm. In some embodiments, a layered structure can have a length of atleast about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. Insome embodiments, a layered structure can have a length of at leastabout 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

The width of a layered structure can be engineered to fit the functionor use of a synthetic leather. A layered structure can have a width fromabout 0.01 mm to about 50 m. For example, a layered structure can have awidth from about 0.01 mm to about 10 mm, from about 0.01 mm to about 8mm, from about 0.01 to about 5 mm, from about 0.02 to about 5 mm, fromabout 0.05 to about 5 mm, from about 0.1 to about 5 mm, from about 0.1to about 2 mm, from about 0.1 to about 1 mm, from about 0.1 to about 0.8mm, or from about 0.1 to about 0.5 mm. For example, a layered structurecan have a width from about 0.02 mm to 5 mm. For example, a layeredstructure can have a width from about 0.1 mm to 0.5 mm. For example, alayered structure can have a width from about 0.2 mm to 0.5 mm. In somecases, the width of a layered structure can be at least 0.001 mm, 0.01mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of a layeredstructure can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01mm. In some embodiments, a layered structure can have a width of atleast about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm.In some embodiments, a layered structure can have a width of at leastabout 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In someembodiments, a layered structure can have a width of at least about 50,60, 70, 80, 90, 100, 200, 300, 400 m.

A layered structure can comprise fibroblasts and keratinocytes at anyratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1,24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2,1:10, or 1:100. In some cases, the ratio of fibroblasts to keratinocytescan be from about 20:1 to about 3:1, from about 20:1 to about 4:1, fromabout 20:1 to about 5:1, from about 20:1 to about 10:1, or from about20:1 to about 15:1.

A layered structure can comprise fibroblasts and melanocytes at anyratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1,24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2,1:10, or 1:100. In some cases, the ratio of fibroblasts to melanocytecan be from about 20:1 to about 3:1, from about 20:1 to about 4:1, fromabout 20:1 to about 5:1, from about 20:1 to about 10:1, or from about20:1 to about 15:1.

A layered structure can comprise keratinocytes and melanocytes at anyratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1,24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2,1:10, or 1:100. In some cases, the ratio of keratinocytes to melanocytecan be from about 20:1 to about 3:1, from about 20:1 to about 4:1, fromabout 20:1 to about 5:1, from about 20:1 to about 10:1, or from about20:1 to about 15:1.

One type of cells in a layered structure can comprise at most 99%, 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 10%, 5%, or 1% of the total cell population in the layeredstructure. One type of cells in a layered structure can comprise aboutat least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the total cell population in the layered structure. Forexample, fibroblasts in a layered structure can comprise about at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%of the total cell population in the layered structure.

Synthetic Leather

A synthetic leather can be formed by one or more layered structures. Forexample, a synthetic leather can be formed by at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 layeredstructures.

A synthetic leather can be of various thickness. For example, asynthetic leather can have a thickness resembling to a natural leather.In some cases, a synthetic leather can have a thickness from about 0.001mm to about 100 mm. For example, a layered structure can have athickness from about 0.005 mm to about 50 mm, from about 0.005 to about10, from about 0.01 mm to about 10 mm, from about 0.1 to about 5 mm,from about 0.5 mm to about 5 mm, from about 0.5 mm to about 3 mm, fromabout 0.8 mm to about 3 mm, from about 0.8 mm to about 2 mm, from about0.8 mm to about 1.8 mm, from about 0.8 mm to about 1.6 mm, from about0.9 mm to about 1.4 mm, from about 1 mm to about 1.5 mm, from about 1 mmto about 1.4 mm, or from about 1 mm to about 1.3 mm. In some cases, thethickness of a synthetic leather can be at least 0.001 mm, 0.01 mm, 0.02mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm,8 mm, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. In some cases, thethickness of a synthetic leather can be at most 100 mm, 50 mm, 40 mm, 20mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some cases, the thickness of asynthetic leather can be about 1.2 mm.

A synthetic leather can have a length from about 0.01 mm to about 50 m.For example, a synthetic leather can have a length from about 0.01 mm toabout 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, fromabout 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 toabout 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about0.5 mm. For example, a synthetic leather can have a length from about0.02 mm to 5 mm. For example, a synthetic leather can have a length fromabout 0.1 mm to 0.5 mm. For example, a synthetic leather can have alength from about 0.2 mm to 0.5 mm. In some cases, the length of asynthetic leather can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm,0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10mm. In some cases, the length of a synthetic leather can be at most 50mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm,0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, asynthetic leather can have a length of at least about 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, asynthetic leather can have a length of at least about 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, asynthetic leather can have a length of at least about 50, 60, 70, 80,90, 100, 200, 300, 400 m.

A synthetic leather can have a width from about 0.01 mm to about 50 m.For example, a synthetic leather can have a width from about 0.01 mm toabout 10 mm, from about 0.01 mm to about 8 mm, from about 0.01 to about5 mm, from about 0.02 to about 5 mm, from about 0.05 to about 5 mm, fromabout 0.1 to about 5 mm, from about 0.1 to about 2 mm, from about 0.1 toabout 1 mm, from about 0.1 to about 0.8 mm, or from about 0.1 to about0.5 mm. For example, a synthetic leather can have a width from about0.02 mm to 5 mm. For example, a synthetic leather can have a width fromabout 0.1 mm to 0.5 mm. For example, a synthetic leather can have awidth from about 0.2 mm to 0.5 mm. In some cases, the width of asynthetic leather can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm,0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10mm. In some cases, the width of a synthetic leather can be at most 50mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm,0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, asynthetic leather can have a width of at least about 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, a syntheticleather can have a width of at least about 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700 cm. In some embodiments, a synthetic leather canhave a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

Basement Membrane Substitute

A synthetic leather can further comprise a basement membrane substitute.A basement membrane substitute can be between two cell layers, e.g.,between a dermal layer and an epidermal layer. A basement membranesubstitute can be a dermo-epidermal junction similar to that whichexists in vivo, from a structural point of view and/or from abiochemical point of view. From the biochemical point of view, abasement membrane substitute can comprise components of the basalmembrane, of the lamina densa, of the lamina lucida and of the sub-basalzone, such as, collagen IV, collagen VII, laminin 5, entactinfibronectin, or any combination thereof.

A basement membrane substitute in a synthetic leather can be urinarybasement membrane (UBM), liver basement membrane (LBM), amnion, chorion,allograft pericardium, allograft acellular dermis, amniotic membrane,Wharton's jelly, or any combination thereof. For example, a basementmembrane substitute can be a dried acellular amniotic membrane. Incertain cases, a basement membrane substitute can be a polymer, e.g., ananopolymer. For example, a basement membrane substitute can benano-fibrous poly hydroxybutyrate-cohydroxyvalerate (PHBV), as describedby Bye et al., Journal of Biomaterials and Tissue Engineering Vol. 4,1-7, 2014.

Scaffold

A cell layer (e.g., a dermal layer), a layered structure, or a syntheticleather can be placed on a scaffold. A scaffold can provide certainfirmness (e.g., resistance to tearing), elasticity, or both. In somecases, a part of or the entire scaffold can be comprise in the syntheticleather. In other cases, a scaffold may not comprised in the syntheticleather. After assisting the formation of a layer in a syntheticleather, a scaffold can be removed from the final synthetic leatherproduct. In certain cases, a scaffold comprised in a synthetic leathercan be degraded after a period of time. A scaffold described herein cancomprise a trabecular pattern.

A scaffold can be made of natural materials, synthetic materials, orcombination thereof. Examples of scaffolds include a scaffold formedusing a net made of a bioabsorbable synthetic polymer, a scaffold formedby attaching a nylon net to a silicon film, a scaffold having atwo-layered structure of a collagen sponge and a silicon sheet, ascaffold formed using an atelo collagen sponge made into a sheet, ascaffold formed by matching collagen sponges having different poresizes, and acellular dermal matrices (ADM) formed using fibrin glue orallogeneic skin that has been made cell-free.

A scaffold can comprise natural substances such as collagen (e.g.,collagen matrix), natural adhesive (e.g., fibrin glue, cold glues,animal glue, blood albumen glue, casein glue, or vegetable glues such asstarch and dextrin glues). In some cases, a scaffold comprises silk. Forexample, a scaffold can be made of silk. In some embodiments, a scaffoldcan comprise, silk fibroin, cellulose, cotton, acetate, acrylic, latexfibers, linen, nylon, rayon, velvet, modacrylic, olefin polyester,saran, vinyon, wool, jute, hemp, bamboo, flax or a combination thereof.In some embodiments, a scaffold can comprise fibers. In someembodiments, the fibers can be fibers of silk, cotton, wool, linen,cellulose extracted in particular from wood, vegetables or algae,polyamide, modified cellulose (rayon, viscose, acetate, especially rayonacetate), poly-p-phenyleneterephthalamide, acrylic fibers, for examplethose of polymethyl methacrylate or of poly-2-hydroxyethyl methacrylate,fibers of polyolefin for example fibers of polyethylene orpolypropylene, glass, silica, aramid, carbon, for example in the form ofgraphite, poly(tetrafluoroethylene), insoluble collagen, polyesters,polyvinyl chloride or polyvinylidene chloride, polyvinyl alcohol,polyacrylonitrile, chitosan, polyurethane, poly(urethane-urea) orpolyethylene phthalate, and fibers formed from a blend of polymers suchas those mentioned above, such as polyamide/polyester fibers or anycombination thereof.

A scaffold can comprise polymers. A polymer can be a biopolymer. Abiopolymer can include but is not limited to chitin, chitosan, elastin,collagen, keratin or polyhydroxyalkanoate. The polymers can bebiodegradable, biostable, or combinations thereof. The polymer in ascaffold can be natural polymers. Exemplary natural polymers includepolysaccharides such as alginate, cellulose, dextran, pullane,polyhyaluronic acid, chitin, poly(3-hydroxyalkanoate),poly(3-hydroxyoctanoate) or poly(3-hydroxyfatty acid). In some cases, ascaffold also comprises chemical derivatives of the natural polymers.Such chemical derivatives can include substitutions and/or additions ofchemical groups such as alkyl, alkylene, hydroxylations, oxidations, aswell as other modifications familiar to those skilled in the art. Thenatural polymers can also be selected from proteins such as collagen,zein, casein, gelatin, gluten, and serum albumen. The polymer in ascaffold can be biodegradable synthetic polymers, including polyalpha-hydroxy acids such as poly L-lactic acid (PLA), polyglycolic acid(PGA) or copolymers thereof (e.g., poly D,L-lactic co-glycolic acid(PLGA)), and hyaluronic acid.

A scaffold can be bioabsorbable. A bioabsorbable scaffold can be anon-cytotoxic structure or substance that can be capable of containingor supporting living cells and holding them in a desired configurationfor a period of time. The term “bioabsorbable” can refer to any materialthe body can break down into non-toxic by-products that are excretedfrom the body or metabolized therein. Exemplary bioabsorbable materialsfor a scaffold include, poly(lactic acid), poly(glycolic acid),poly(trimethylene carbonate), poly(dimethyltrimethylene carbonate),poly(amino acids)s, tyrosine-derived poly(carbonates)s,poly(carbonates)s, poly(caprolactone), poly(para-dioxanone),poly(esters)s, poly(ester-amides)s, poly(anhydrides)s, poly(orthoesters)s, collagen, gelatin, serum albumin, proteins, polysaccharides,mucopolysaccharides, carbohydrates, glycosaminoglycans, poly(ethyleneglycols)s, poly(propylene glycols)s, poly(acrylate esters)s,poly(methacrylate esters)s, poly(vinyl alcohol), hyaluronic acid,chondroitin sulfate, heparin, dermatan sulfate, versican, copolymers,blends and mixtures of polymers, and oligomers containing bioabsorbablelinkages.

A scaffold can be of various thicknesses. For example, a scaffold canhave a thickness that can be suitable for forming a cell layer. Forexample, a scaffold can have a thickness from about 0.1 mm to about 10mm, such as from about 0.1 mm to about 5 mm, from about 0.1 mm to about4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm,to about 0.1 mm to about 1 mm, from about 0.2 mm to about 1 mm, fromabout 0.3 mm to about 1 mm, from about 0.4 mm to about 1 mm, from about0.5 mm to about 1 mm, from 0.3 mm to about 1.5 mm, from about 0.4 mm toabout 1.2 mm, from about 0.6 mm to about 1.2 mm, or from about 0.7 mm toabout 1.5 mm. For example, a scaffold can have a thickness from about0.5 mm to lmm. In some cases, a scaffold can be at least 0.1 mm, 0.2 mm,0.3 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm thick.In some cases, a scaffold can be at most 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm thick. In someembodiments, a scaffold can have a length and/or a width of a cell layerto be placed and/or grown upon a scaffold. In some embodiments, ascaffold can have a length and/or a width of a cell layer describedherein.

A scaffold can have a surface area on a face of a synthetic leather. Forexample, a scaffold can have a surface area of from about 0.1 mm² toabout 100 mm², from about 0.1 mm² to about 95 mm², from about 0.1 mm² toabout 90 mm², from about 0.1 mm² to about 85 mm², from about 0.1 mm² toabout 80 mm², from about 0.1 mm² to about 75 mm², from about 0.1 mm² toabout 70 mm², from about 0.1 mm² to about 65 mm², from about 0.1 mm² toabout 60 mm², from about 0.1 mm² to about 55 mm², from about 0.1 mm² toabout 50 mm², from about 0.1 mm² to about 45 mm², from about 0.1 mm² toabout 40 mm², from about 0.1 mm² to about 35 mm², from about 0.1 mm² toabout 30 mm², from about 0.1 mm² to about 25 mm², from about 0.1 mm² toabout 20 mm², from about 0.1 mm² to about 15 mm², from about 0.1 mm² toabout 10 mm², from about 0.1 mm² to about 5 mm², or from about 0.1 mm²to about 1 mm². In some cases, a scaffold can have a surface area offrom about 0.1 cm² to about 100 cm², from about 0.1 cm² to about 95 cm²,from about 0.1 cm² to about 90 cm², from about 0.1 cm² to about 85 cm²,from about 0.1 cm² to about 80 cm², from about 0.1 cm² to about 75 cm²,from about 0.1 cm² to about 70 cm², from about 0.1 cm² to about 65 cm²,from about 0.1 cm² to about 60 cm², from about 0.1 cm² to about 55 cm²,from about 0.1 cm² to about 50 cm², from about 0.1 cm² to about 45 cm²,from about 0.1 cm² to about 40 cm², from about 0.1 cm² to about 35 cm²,from about 0.1 cm² to about 30 cm², from about 0.1 cm² to about 25 cm²,from about 0.1 cm² to about 20 cm², from about 0.1 cm² to about 15 cm²,from about 0.1 cm² to about 10 cm², from about 0.1 cm² to about 5 cm²,or from about 0.1 cm² to about 1 cm². In some cases, a scaffold can havea surface area of from about 0.1 m² to about 100 m², from about 0.1 m²to about 95 m², from about 0.1 m² to about 90 m², from about 0.1 m² toabout 85 m², from about 0.1 m² to about 80 m², from about 0.1 m² toabout 75 m², from about 0.1 m² to about 70 m², from about 0.1 m² toabout 65 m², from about 0.1 m² to about 60 m², from about 0.1 m² toabout 55 m², from about 0.1 m² to about 50 m², from about 0.1 m² toabout 45 m², from about 0.1 m² to about 40 m², from about 0.1 m² toabout 35 m², from about 0.1 m² to about 30 m², from about 0.1 m² toabout 25 m², from about 0.1 m² to about 20 m², from about 0.1 m² toabout 15 m², from about 0.1 m² to about 10 m², from about 0.1 m² toabout 5 m², or from about 0.1 m² to about 1 m².

In some cases, a scaffold can have a surface area of at least about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 75, or 100 mm². In some cases, a scaffold can have asurface area of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 cm². Insome cases, a scaffold can have a surface area of at least about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 75, or 100 m².

Alternatively, a cell layer may not form on a scaffold. For example, adermal layer may not form on a scaffold (e.g., collagen matrix). Incertain cases, a synthetic leather does not comprise a scaffold.

Pigments

A synthetic leather can comprise one or more pigments. One or more layerstructures of the synthetic leather can be pigmented. A pigment in asynthetic leather can be a natural pigment produced in cells forming thesynthetic leather. For example, a pigment can be melanin, includingeumelanin (e.g., brown eumelanin and black eumelanin), pheomelanin,neuromelanin, or any combination thereof. A pigment in a syntheticleather can be an exogenous pigment, such as a leather pigment dye.

Collagen

A synthetic leather can comprise collagen. Collagen can refer to anymember of a family of at least 28 distinct collagen types. Collagens canbe characterized by a repeating triplet of amino acids, -(Gly-X-Y)n-, sothat approximately one-third of the amino acid residues are in collagenare glycine. X can be proline and Y can be hydroxyproline. Thus, thestructure of collagen can have twined triple units of peptide chains ofdiffering lengths. A synthetic leather can comprise collagen from one ormore species. In some cases, a synthetic leather comprises collagen fromdifferent animals. Different animals can produce different amino acidcompositions of the collagen, which can result in different properties(and differences in the resulting leather). Collagen fiber monomers canbe produced from alpha-chains of about 1050 amino acids long, so thatthe triple helix takes the form of a rod of about 300 nm long, with adiameter of about 1.5 nm.

A synthetic leather can comprise one or more types of collagen. Collagencomprised in a synthetic leather can include fibrillary collagens,non-fibrillar collagens, or a combination thereof. Fibrillary collagensinclude type I, type II, type III, type V, and type XI collagens.Non-fibrillar collagens include fibril associated collagens withinterrupted triple helices (e.g., type IX, type XII, type XIV, type XVI,and type XIX), short chain collagens (e.g., type VIII and type X),basement membrane collagens (type IV), Multiplexin (Multiple TripleHelix domains with Interruptions) (e.g., Type XV and type XVIII), MACITcollagens (Membrane Associated Collagens with Interrupted TripleHelices) (e.g., Type XIII and type XVII).

Collagen can be comprised in one or more parts of a synthetic leather.For example, collagens can be comprised in one or more dermal layers,one or more epidermal layers, or combination thereof, in a syntheticleather. For example, collagens can be comprised in one or more layeredstructures in a synthetic leather. In some cases, when part of thesynthetic leather can be removed during process, collagen can also becomprised in the removed product.

Collagen in a synthetic leather can be from one or more sources. Forexample, the collagen can be produced by collagen producing cells in thesynthetic leather. For example, the collagen can be separately added tothe leather. In some cases, a synthetic leather comprises collagenproduced by collagen producing cells and collagens separately added.

At least part of the collagen in a synthetic leather can be produced bycollagen producing cells. Such collagen producing cells can be comprisedin the synthetic leather. Exemplary collagen producing cells includeepithelial cells, fibroblasts, keratinocytes, comeocytes, melanocytes,Langerhans cells, basal cells, smooth muscle cells, or a combinationthereof. The epithelial cells can include squamous cells, cuboidalcells, columnar cells, basal cells, or a combination thereof. Thefibroblasts can include dermal fibroblasts. The keratinocytes caninclude epithelial keratinocytes, basal keratinocytes, proliferatingbasal keratinocytes, differentiated suprabasal keratinocytes, or acombination thereof. Collagen in a synthetic leather can be produced byone or more types of collagen-producing cells.

Additives

A synthetic leather can further comprise one or more additives. Suchadditives can enhance the commercial appeal (e.g., appearance, color, orodor). Exemplary additives include minerals, fiber, fatty acids, andamino acids, proteins. An additive can be an odorant.

Additives can include one or more of: matrix proteins, proteoglycans,antioxidants, perfluorocarbons, and growth factors. A growth factor canbe a protein, a polypeptide, or a complex of polypeptides, includingcytokines (e.g., that are produced by a cell and which can affect itselfand/or a variety of other neighboring or distant cells). Growth factorscan affect the growth and/or differentiation of specific types of cells,either developmentally or in response to a multitude of physiological orenvironmental stimuli. Some, but not all, growth factors are hormones.Exemplary growth factors include insulin, insulin-like growth factor(IGF), nerve growth factor (NGF), vascular endothelial growth factor(VEGF), keratinocyte growth factor (KGF), fibroblast growth factors(FGFs), including basic FGF (bFGF), platelet-derived growth factors(PDGFs), including PDGF-AA and PDGF-AB, hepatocyte growth factor (HGF),transforming growth factor alpha (TGF-a), transforming growth factorbeta (TGF-β), including TGFpi and TGFP3, epidermal growth factor (EGF),granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocytecolony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, and thelike. Other polypeptides or molecules (e.g., healing agents; enzymessuch as matrix-degrading enzymes and matrix-degrading enzyme inhibitors(e.g., TIMPs), antibiotics, and antimycotics) can also be added to asynthetic leather.

Additives can also include preservatives known to the art. Exemplarypreservatives include antimicrobial preservatives such as calciumpropionate, sodium nitrate, sodium nitrite, sulfites (e.g., sulfurdioxide, sodium bisulfate, potassium hydrogen sulfite, etc.), disodiumethylenediammetetraacetic acid (EDTA), antioxidant such as butylatedhydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

In certain cases, a synthetic leather can comprise an extracellularmatrix or connective tissue. For example, a synthetic leather canfurther comprise collagen, keratin, elastin, gelatin, proteoglycan,dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin,dermatopontin, lipid, fatty acid, carbohydrate, and a combinationthereof.

Pattern of Synthetic Leather

A synthetic leather can be patterned. For example, the synthetic leathermay be patterned after a skin pattern of an animal selected fromantelope, bear, beaver, bison, boar, camel, caribou, cat, cattle, deer,dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo,lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, seal, sheep,squirrel, tiger, whale, wolf, yak, zebra, turtle, snake, crocodile,alligator, dinosaur, frog, toad, salamander, newt, chicken, duck, emu,goose, grouse, ostrich, pheasant, pigeon, quail, turkey, anchovy, bass,catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut,herring, mackerel, mahi mahi, manta ray, marlin, orange roughy, perch,pike, pollock, salmon, sardine, shark, snapper, sole, stingray,swordfish, tilapia, trout, tuna, walleye, and a combination thereof. Thepattern can be a skin pattern of a fantasy animal selected from dragon,unicorn, griffin, siren, phoenix, sphinx, Cyclops, satyr, Medusa,Pegasus, Cerberus, Typhoeus, gorgon, Charybdis, empusa, chimera,Minotaur, Cetus, hydra, centaur, fairy, mermaid, Loch Ness monster,Sasquatch, thunderbird, yeti, chupacabra, and a combination thereof.

A synthetic leather can be made to resemble traditional animal skin,hide, or leather products and design parameters (e.g., cell types,additives, size, shape). In some cases, a synthetic leather comprises acell layer characterized by a composition that can be substantiallysimilar to traditional animal skin, hide, or leather products. Forexample, such layer can be characterized by a composition that can besubstantially about 60% to 80% aqueous fluid, about 14%-35% protein,about 1%-25% fat. In some cases, keratinocytes of the cell layer arealigned. For example, the keratinocytes can be aligned by application ofan electrical field. For example, keratinocytes can be aligned byapplication of a mechanical stimulus, such as cyclical stretching andrelaxing the substratum. In some cases, aligned (e.g., electro-orientedand mechano-oriented) keratinocytes have substantially the sameorientation with regard to each other as can be found in many animalskin tissues.

Leather Articles

A synthetic leather herein can be at least a portion of a leatherarticle. For example, a synthetic leather can be used as substitute ofnatural leather in a leather article. Exemplary leather articles includea watch strap, belt, suspender, packaging, shoe, boot, footwear, glove,clothing (e.g., tops, bottoms, and outerwear), luggage, bag (e.g., ahandbag with or without shoulder strap), clutch, purse, coin purse,billfold, key pouche, credit card case, pen case, backpack, cases,wallet, saddle, harness, whip, travel goods (e.g., a trunk, suitcase,travel bag, beauty case, or a toilet kit), rucksacks, portfolio,document bag, briefcase, attache case, pet article (e.g., a leash orcollar), hunting and fishing article (e.g., a gun case, cutlery case, ora holster for firm arms), a stationary article (e.g., a writing pad,book cover, camera case, spectacle case, cigarette case, cigar case,jewel case, or a mobile phone holster), a sport article (e.g., a ballsuch as basketball, soccer ball, or a football), a building interior, abuilding exterior, an upholstery, a book binding, a furniture, a lamp, alamp shade, a table covering, a wall covering, a floor covering, aceiling covering, a car interior, a car exterior, a boat interior, aboat exterior, an airplane interior, a yacht interior, a yacht exterior,a pillow case, a sheet, a duvet cover, jewelry, an accessory, a pair ofglasses, a pair of sun glasses, or a consumer electronic. For example, aleather article can be a watch wrap. For example, a leather article canbe a belt. For example, a leather article can be a bag.

Skin Graft

A synthetic leather or portions thereof can also be used as a skingraft, e.g., an allograft or xenograft for transplanting to a subject.For example, the synthetic leather, dermal layer, epidermal layer and/ora layered structure can be a source of skin graft for allotransplant orxenotransplant. In some cases, the synthetic leather, dermal layer,epidermal layer and/or a layered structure can be produced with cellsgenetically modified to reduce immune-rejection in the recipient of thegraft.

Methods

Also disclosed herein are methods of making a synthetic leather. Themethods can comprise forming an artificial dermal layer, forming anartificial epidermal layer, or a combination thereof. The methods canfurther comprise tanning at least of a portion of the artificial dermallayer and/or artificial epidermal layer. The cells in a syntheticleather, e.g., those in the dermal layer and/or the epidermal layer canbe differentiated from stem cells (e.g., iPSCs). The methods herein canfurther comprise differentiating stem cells (e.g., iPSCs) into cells inthe synthetic leather, e.g., cells in the dermal layer and/or theepidermal layer. In certain cases, the methods comprise placing a firstcell layer (e.g., an epidermal layer) upon a second cell layer (e.g., adermal layer) thereby forming a layered structure, and tanning at leasta portion of the layered structure. In some cases, the methods canfurther comprise removing at least a portion of the first cell layer(e.g., an epidermal layer).

Forming Cell Layers

A cell layer can be formed by preparing a plurality of multicellularbodies comprising one or more type of cells, and arranging suchmulticellular bodies to form a cell layer. For example, a cell layer canbe formed by adjacently arranging a plurality of multicellular bodies,wherein the multicellular bodies are fused to form a planar layer.

Forming a cell layer may need a scaffold. A cell layer can be formed byarranging a plurality of multicellular bodies on a scaffold. Forexample, the forming step can comprise arranging or placingmulticellular bodies on a support substrate that allows themulticellular bodies to fuse to form a layer (e.g., a substantiallyplanar layer). In some cases, the multicellular bodies or the layers arearranged horizontally and/or vertically adjacent to one another.Alternatively, forming a cell layer may not need a scaffold.

Cell layers can be formed by embedding cells in a medium or gel. In somecases, dermal layers can be formed using fibroblasts embedded in acollagen I or fibrin gel. Other types of media can also be used. Forexample, a medium can promote fibroblast to secret sufficient amount ofextracellular matrix to enable extended maintenance of epidermis withoutthe need for collagen gels.

Forming Multicellular Bodies

There are various ways to make multicellular bodies having thecharacteristics described herein. In some cases, a multicellular bodycan be fabricated from a cell paste containing a plurality of cells,e.g., with a desired cell density and viscosity. In further cases, thecell paste can be shaped into a desired shape and a multicellular bodyformed through maturation (e.g., incubation). In some cases, an elongatemulticellular body can be produced by shaping a cell paste including aplurality of cells into an elongate shape (e.g., a cylinder). In furthercases, the cell paste can be incubated in a controlled environment toallow the cells to adhere and/or cohere to one another to form theelongate multicellular body. For example, a multicellular body can beproduced by shaping a cell paste including a plurality of living cellsin a device that holds the cell paste in a three-dimensional shape. Insome cases, the cell paste can be incubated in a controlled environmentwhile it can be held in the three dimensional shape for a sufficienttime to produce a body that has sufficient cohesion to support itself ona flat surface, as described herein.

A cell paste can be provided by: (A) mixing cells or cell aggregates (ofone or more cell types) and a cell culture medium (e.g., in apre-determined ratio) to result in a cell suspension, and (B) compactingthe cellular suspension to produce a cell paste with a desired celldensity and viscosity. Compacting can be achieved by a number ofmethods, such as by concentrating a particular cell suspension thatresulted from cell culture to achieve the desired cell concentration(density), viscosity, and consistency required for the cell paste. Insome cases, a relatively dilute cell suspension from cell culture can becentrifuged for a determined time to achieve a cell concentration in thepellet that allows shaping in a mold. Tangential flow filtration (“TFF”)is another suitable method of concentrating or compacting the cells. Insome cases, compounds are combined with the cell suspension to lend theextrusion properties required. Suitable compounds include, collagen,hydrogels, Matrigel, nanofibers, self-assembling nanofibers, gelatin,and fibrinogen. One or more ECM components (or derivatives of ECMcomponents) can also be included by, resuspending the cell pellet in oneor more physiologically acceptable buffers containing the ECM components(or derivatives of ECM components) and the resulting cell suspensioncentrifuged again to form the cell paste.

Various methods can be used to shape the cell paste. For example, in aparticular embodiment, the cell paste can be manually molded or pressed(e.g., after concentration/compaction) to achieve a desired shape. Byway of a further example, the cell paste can be taken up (e.g.,aspirated) into a preformed instrument, such as a micropipette (e.g., acapillary pipette), that shapes the cell paste to conform to an interiorsurface of the instrument. The cross sectional shape of the micropipette(e.g., capillary pipette) can be alternatively circular, square,rectangular, triangular, or other non-circular cross-sectional shape. Insome embodiments, the cell paste can be shaped by depositing it into apreformed mold, such as a plastic mold, metal mold, or a gel mold. Insome embodiments, centrifugal casting or continuous casting can be usedto shape the cell paste.

The cell paste can be further matured. In some cases, the cell paste canbe incubated at about 37° C. for a time period (which can be cell-typedependent) to foster adherence and/or coherence. Alternatively or inaddition, the cell paste can be held in the presence of cell culturemedium containing factors and/or ions to foster adherence and/orcoherence.

Arranging Multicellular Bodies on a Support Substrate to Form Layers

Multicellular bodies can be arranged on a support substrate to produce adesired three-dimensional structure (e.g., a substantially planarlayer). For example, multicellular bodies can be manually placed incontact with one another, deposited in place by extrusion from apipette, nozzle, or needle, or positioned in contact by an automatedmachine such as a biofabricator.

A support substrate can be permeable to fluids, gasses, and nutrientsand allows cell culture media to contact all surfaces of themulticellular bodies and/or layers during arrangement and subsequentfusion. In some cases, a support substrate can be made from naturalbiomaterials such as collagen, fibronectin, laminin, and otherextracellular matrices. In some cases, a support substrate can be madefrom synthetic biomaterials such as hydroxyapatite, alginate, agarose,polyglycolic acid, polylactic acid, and their copolymers. In some cases,a support substrate can be solid, semisolid, or a combination of solidand semisolid support elements. In some cases, a support substrate canbe planar to facilitate production of planar layers. In some cases, asupport substrate can be raised or elevated above a non-permeablesurface, such as a portion of a cell culture environment (e.g., a Petridish, a cell culture flask, etc.) or a bioreactor. A permeable, elevatedsupport substrate can contribute to prevention of premature cell death,contributes to enhancement of cell growth, and facilitates fusion ofmulticellular bodies to form layers.

Once assembly of a layer is complete, a tissue culture medium can bepoured over the top of the construct. In some cases, the tissue culturemedium enters the spaces between the multicellular bodies to support thecells in the multicellular bodies. The multicellular bodies in thethree-dimensional construct can be allowed to fuse to one another toproduce a layer (e.g., a substantially planar) for use in formation ofthe synthetic leather. The terms “fuse,” “fused” or “fusion,” can meanthat the cells of contiguous multicellular bodies become adhered and/orcohered to one another, either directly through interactions betweencell surface proteins, or indirectly through interactions of the cellswith ECM components or derivatives of ECM components. A fused layer canbe completely fused and that multicellular bodies have becomesubstantially contiguous. Alternatively, a fused layer can besubstantially fused or partially fused and the cells of themulticellular bodies have become adhered and/or cohered to the extentnecessary to allow moving and manipulating the layer intact.

Multicellular bodies can fuse to form a layer in a cell cultureenvironment (e.g., a Petri dish, cell culture flask, or bioreactor). Insome cases, the multicellular bodies fuse to form a layer in anenvironment with conditions suitable to facilitate growth of the celltypes included in the multicellular bodies. In some cases, fusing takesplace over about 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, andincrements therein. In other cases, fusing takes place over about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, and 48 hours, and increments therein. In yetother cases, fusing takes place over about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, and 14 days, and increments therein. In further cases, fusingtakes place over about 2 hours to about 24 hours. Factors relevant tothe fusing time can include cell types, cell type ratios, cultureconditions, and the presence of additives such as growth factors.

Once fusion of a layer is complete, the layer and the support substratecan be separated. In other cases, the layer and the support substrateare separated when fusion of a layer is substantially complete orpartially complete, but the cells of the layer are adhered and/orcohered to one another to the extent necessary to allow moving,manipulating, and stacking the layer without breaking it apart. Thelayer and the support substrate can be separated via standard proceduresfor melting, dissolving, or degrading the support substrate. In somecases, the support substrate can be dissolved, for example, bytemperature change, light, or other stimuli that do not adversely affectthe layer. In certain cases, the support substrate can be made of aflexible material and peeled away from the layer. The separated layercan be transferred to a bioreactor for further maturation. In somecases, the separated layer matures and further fuses after incorporationinto an engineered animal skin, hide, or leather product.

Alternatively, the layer and the support substrate may not be separated.The support substrate degrades or biodegrades prior to packaging,freezing, sale or consumption of the assembled engineered animal skin,hide, or leather product.

A cell layer can be formed over a period of time. In some cases, a celllayer, e.g., an epidermal layer or a dermal layer, can be formed within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, 60, 120, 300 days. In some cases, a dermal layer can beformed in about 1 to 15 days, e.g., 5 to 10 days, or 10 to 12 days. Insome cases, a dermal layer can be formed about 5 to 25 days, e.g., 14 to15 days.

The present disclosure provides methods for making synthetic leatherimproved barrier function. In some cases, the methods comprise providingkeratinocytes and a culture media comprising ascorbic acid and linoleicacid; and culturing the keratinocytes under conditions such that asynthetic leather having improved barrier function can be formed. Insome cases, the culture conditions include culture at about 50 to 95%humidity, e.g., about 75% humidity. In some cases, the ascorbic acid canbe provided at concentration of from about 10 to 100 micrograms/ml. Instill further cases, linoleic acid can be provided at a concentration offrom about 5 to 80 micromolar. The present disclosure is not limited tosynthetic leather formed from a particular source of keratinocytes.Indeed, the synthetic leather can be formed from a variety of primaryand immortal keratinocytes, including, but not limited to Near-DiploidImmortalized Keratinocytes (NIKS) cells. In still further cases, thekeratinocytes express exogenous wild-type or variant Kruppel-like factor(GKLF). In still further cases, the keratinocytes are derived from twodifferent sources. In other cases, the synthetic leather has a surfaceelectrical capacitance of from about 40 to about 240 pF. In somepreferred cases, the skin equivalent has a surface electricalcapacitance of from about 80 to about 120 pF. In other preferred cases,the content of ceramides 5, 6, and 7 in the skin equivalent can be fromabout 20 to about 50% of total ceramide content. In still otherpreferred cases, the content of ceramide 2 in the skin equivalent can befrom about 10 to about 40% of total ceramide content. In still furthercases, the present disclosure provides the skin equivalent made by themethod just described.

Arranging Layers to Form a Layered Structure

Multiple cell layers can be arranged to form a layer structure, thusproducing synthetic leathers described herein. In some cases, dermallayers and epidermal layers are formed separately and assembled byplacing the epidermal layers atop of the dermal layers (e.g., when bothan epidermal layer and a dermal layer are fully formed). In some cases,an epidermal layer can be grown atop a dermal layer. In certain cases, abasement membrane or basement membrane substitute can be placed betweena dermal layer and an epidermal layer. For example, the cell layers canbe manually placed in contact with one another or deposited in place byan automated, computer-aided machine such as a biofabricator, accordingto a computer script.

Before assembling multiple cell layers, one or more quality controlsteps can be performed. For example, Trans Epithelial ElectricalResistance (TEER) can be performed on epidermis before placement ondermis (e.g., 0 day), followed by histology analysis (e.g., minimum 3˜5days). Using methods provided herein, the risk of improperly formedlayered structure or full thickness skin equivalents can be low.

Multiple cell layers can be assembled in various ways. In some cases, anepidermal layer and a dermal layer (with or without a basement membranesubstitute) are placed on a scaffold (e.g., silk), e.g., to achievethickness and tensile strength of natural leather. In some cases, anepidermal layer and multiple dermal layers (with or without a basementmembrane substitute) are assembled without using a scaffold. Suchassembly can achieve thickness and tensile strength that resemblenatural leather. In some cases, an epidermal layer and multiple dermallayers (with or without a basement membrane substitute) are placed on ascaffold (e.g., silk) achieve thickness and tensile strength thatresemble natural leather.

In some embodiments, chemical, mechanical, performance, strength,durability, moisture, dimensional tests or a combination thereof can beperformed on one or more multiple cell layer, synthetic leathers,artificial epidermal layers, artificial dermal layers, layeredstructures, products produced therefrom. In some embodiments, achemical, mechanical, performance, strength, durability, moisture,dimensional tests or a combination thereof can be performed using anon-standard test. In some embodiments, a chemical, mechanical,performance, strength, durability, moisture, dimensional tests or acombination thereof can be performed using a standard test. In someembodiments, a test can be performed as instructed and/or adopted and/orratified and/or developed by the International Standards Organization(ISO), European standards body (CEN), ASTM International or by theInternational Union of Leather Technicians and Chemists (IULTCS). Insome embodiments, a test in any one of Table 1-Table 11 or any variationthereof can be performed using any one or more corresponding method orany variation thereof.

TABLE 1 IULTCS - CHEMICAL TEST METHODS IU No. Method name IUC 1 Generalcomments IUC 2 Sampling IUC 3 Preparation of test material by grindingIUC 4 Determination of substances (fats and other soluble) soluble inDichloromethane. IUC 5 Determination of volatile matter IUC 6Determination of water soluble matter, water soluble inorganic matterand water soluble organic matter IUC 7 Determination of sulphated totalash and sulphated water insoluble ash IUC 8 Determination of chromicoxide IUC 9 Determination of water soluble magnesium salts IUC 10Determination of nitrogen and hide substance IUC 11 Determination of pHand difference figure IUC 13 Determination of zirconium IUC 15Determination of phosphorus IUC 16 Determination of aluminium IUC 17Determination of hydroxyproline in materials containing collagen IUC 18Photometric Determination of chromium (VI) using 1,5-DiphenylcarbazideIUC 19 Determination of formaldehyde content of leather IUC 20 Methodfor the detection of certain AZO colourants in dyed leather IUC 21Method for the detection of certain AZO colourants in dyestuff mixturesIUC 22 Determination of aluminium oxide content of aluminium tanningagents IUC 23 Determination of the pH of aqueous solutions of aluminiumtanning agents IUC 24 Determination of basicity of aluminium tanningagents. IUC 25 Determination of pentachlorophenol content

TABLE 2 IULTCS - PHYSICAL TEST METHODS IU No. Method name IUP 1 Generalremarks IUP 2 Sampling IUP 3 Conditioning IUP 4 Measurement of thicknessIUP 5 Measurement of apparent density IUP 6 Measurement of tensilestrength and percentage elongation IUP 7 Measurement of staticabsorption of water IUP 8 Measurement of tear load - Double edge tearIUP 9 Measurement of distension and strength of grain by the Ball BurstTest IUP 10 Water resistance of flexible leather IUP 11 Measurement ofwater resistance of heavy leather IUP 12 Measurement of resistance tograin cracking and the grain crack index IUP 13 Measurement of twodimensional extension IUP 14 Measurement of waterproofness of glovingleathers IUP 15 Measurement of water vapour permeability IUP 16Measurement of shrinkage temperature up to 100° C. IUP 17 Assessment ofthe resistance of air dry insole leathers to heat IUP 18 Resistance ofair dry lining leathers to heat IUP 19 Resistance of air dry upperleather to heat IUP 20 Measurement of flex resistance by flexometermethod IUP 21 Measurement of set in lasting IUP 22 Assessment of scuffdamage by use of the viewing box IUP 23 Measurement of scuff damage IUP24 Measurement of surface shrinkage by immersion in boiling water IUP 26Measurement of resistance to abrasion of heavy leather IUP 28Measurement of the resistance to bending of heavy leather IUP 29Measurement of cold crack temperature of surface coatings IUP 30Measurement of water vapour absorption and desorption (See IUP 42) IUP32 Measurement of area IUP 35 Measurement of dry heat resistance ofleather IUP 36 Measurement of leather softness Draft IUP 37 Measurementof water repellancy of garment leather IUP 38 Measurement of heatresistance of patent leather IUP 39 Measurement of flex resistance bythe vamp flex method IUP 40 Measurement of tear load - Single edge tearIUP 41 Measurement of surface coating thickness IUP 42 Measurement ofwater vapour absorption IUP 43 Measurement of extension set IUP 44Measurement of stitch tear resistance Draft IUP 45 Measurement of waterpenetration pressure Draft IUP 46 Measurement of fogging characteristicsDraft IUP 47 Measurement of resistance to horizontal spread of flameDraft IUP 48 Measurement of abrasion resistance of upholstery leatherDevelopment Measurement of bagginess (IUP 49) Development Measurement ofsoiling (IUP 50) Development Measurement of Surface Friction (IUP 51)Development Measurement of Compressibility (IUP 52)

TABLE 3 IULTCS - FASTNESS TEST METHODS IU No. Method name IUF 105Numbering code for fastness tests IUF 120 Principles of colour fastnesstesting IUF 131 Grey scale for assessing change in colour IUF 132 Greyscale for assessing staining IUF 151 Preparation of Standard StorableChrome leather IUF 201 Approx. determination of solubility of leatherdyes IUF 202 Fastness to acid of dye solutions IUF 203 Stability to acidof dye solutions IUF 205 Stability to hardness of dye solutions IUF 401Fastness to daylight IUF 402 Fastness to light (Xenon arc) IUF 420Fastness to water spotting IUF 421 Fastness to water IUF 423 Fastness towashing IUF 424 Fastness to formaldehyde IUF 426 Fastness toperspiration IUF 434 Fastness to dry-cleaning of small samples IUF 435Fastness to machine washing IUF 441 Fastness in respect to staining rawcrepe rubber IUF 442 Fastness in respect of staining plasticised PVC IUF450 Fastness to and fro rubbing IUF 454 Fastness to buffing of dyedleather IUF 458 Fastness to ironing IUF 470 Adhesion of finish IUF 412Change of colour with accelerated ageing

TABLE 4 ASTM's Leather Standards-Apparel Test Title Designation TestTitle D1913 - 00(2015) Standard Test Method for Resistance to Wetting ofGarment-Type Leathers (Spray Test) D2096 - 11 Standard Test Method forColorfastness and Transfer of Color in the Washing of Leather D2821 - 14Standard Test Method for Measuring the Relative Stiffness of Leather byMeans of a Torsional Wire Apparatus D5053 - 03(2015) Standard TestMethod for Colorfastness of Crocking of Leather D5552 - 10(2015)Standard Test Method for Resistance of Colored Leather to BleedingD6012 - 03(2013) Standard Test Method for Determination of Resistance ofLeather to (Bleeding) Color Stain Transfer D6013 - 00(2010) StandardTest Method for Determination of Area Stability of Leather to LaunderingD6014 - 00(2015) Standard Test Method for Determination of Dynamic WaterAbsorption of Leather Surfaces

TABLE 5 ASTM's Leather Standards-Chemical Analysis Designation TestTitle D2617 - 12 Standard Test Method for Total Ash in Leather D2807 -93(2015) Standard Test Method for Chromic Oxide in Leather (PerchloricAcid Oxidation) D2810 - 13 Standard Test Method for pH of LeatherD2868 - 10(2015) Standard Test Method for Nitrogen Content (Kjeldahl)and Hide Substance Content of Leather, Wet Blue and Wet White D3495 -10(2015) Standard Test Method for Hexane Extraction of Leather D3790 -79(2012) Standard Test Method for Volatile Matter (Moisture) of Leatherby Oven Drying D3897 - 91(2012) Standard Practice for Calculation ofBasicity of Chrome Tanning Liquors D3898 - 93(2015) Standard Test Methodfor Chromic Oxide in Basic Chromium Tanning Liquors D3913 - 03(2015)Standard Test Method for Acidity in Basic Chromium Tanning LiquorsD4653 - 87(2015) Standard Test Method for Total Chlorides in LeatherD4654 - 87(2015) Standard Test Method for Sulfate Basicity in LeatherD4655 - 95(2012) Standard Test Methods for Sulfates in Leather (Total,Neutral, and Combined Acid) D4906 - 95(2012) Standard Test Method forTotal Solids and Ash Content in Leather Finishing Materials D4907 -10(2015) Standard Test Method for Nitrocellulose in Finish on LeatherD5356 - 10(2015) Standard Test Method for pH of Chrome Tanning SolutionsD6016 - 06(2012) Standard Test Method for Determination of Nitrogen,Water Extractable in Leather D6017 - Standard Test Method forDetermination of Magnesium Sulfate (Epsom Salt) in Leather 97(2015)D6018 - 96(2012) Standard Test Method for Determining the Presence ofLead Salts in Leather D6019 - 15 Test Method for Determination ofChromic Oxide in Basic Chromium Tanning Liquors (Ammonium PersulfateOxidation)

TABLE 6 ASTM's Leather Standards-Fats and Oils Designation Test TitleD5346 - 93(2009) Standard Test Method for Determination of the PourPoint of Petroleum Oil Used in Fatliquors and Softening CompoundsD5347 - 95(2012) Standard Test Method for Determination of the AshContent of Fats and Oils D5348 - 95(2012) Standard Test Method forDetermination of the Moisture Content of Sulfonated and Sulfated Oils byDistillation with Xylene D5349 - 95(2012) Standard Test Method forDetermination of the Moisture and Volatile Content of Sulfonated andSulfated Oils by Hot-Plate Method D5350 - 95(2012) Standard Test Methodfor Determination of Organically Combined Sulfuric Anhydride byTitration, Test Method A D5351 - 93(2009) Standard Test Method forDetermination of Organically Combined Sulfuric Anhydride by ExtractionTitration, Test Method B D5352 - 95(2012) Standard Test Method forDetermination of Organically Combined Sulfuric Anhydride Ash-Gravimetric, Test Method C D5353 - 95(2012) Standard Test Method forDetermination of Total Desulfated Fatty Matter D5354 - 95(2012) StandardTest Method for Determination of Total Active Ingredients in Sulfonatedand Sulfated Oils D5355 - 95(2012) Standard Test Method for SpecificGravity of Oils and Liquid Fats D5439 - 95(2012) Standard Test Methodfor Determination of Sediment in Moellon D5440 - 93(2009) Standard TestMethod for Determining the Melting Point of Fats and Oils D5551 -95(2012) Standard Test Method for Determination of the Cloud Point ofOil D5553 - 95(2012) Standard Test Method for Determination of theUnsaponifiable Nonvolatile Matter in Sulfated Oils D5554 - 15 StandardTest Method for Determination of the Iodine Value of Fats and OilsD5555 - 95(2011) Standard Test Method for Determination of Free FattyAcids Contained in Animal, Marine, and Vegetable Fats and Oils Used inFat Liquors and Stuffing Compounds D5556 - 95(2011) Standard Test Methodfor Determination of the Moisture and Other Volatile Matter Contained inFats and Oils Used in Fat Liquors and Softening Compounds D5557 -95(2011) Standard Test Method for Determination of Insoluble ImpuritiesContained in Fats and Oils Used in Fat Liquors and Stuffing CompoundsD5558 - 95(2011) Standard Test Method for Determination of theSaponification Value of Fats and Oils D5559 - 95(2011) Standard TestMethod for Determination of Acidity as Free Fatty Acids/Acid Number inthe Absence of Ammonium or Triethanolamine Soaps in Sulfonated andSulfated Oils D5560 - 95(2011) Standard Test Method for Determination ofNeutral Fatty Matter Contained in Fats and Oils D5562 - 95(2011)Standard Test Method for Determination of the Acidity as Free FattyAcids/Acid Number in the Presence of Ammonium or Triethanolamine SoapsD5564 - 95(2011) Standard Test Method for Determination of the TotalAmmonia Contained in Sulfonated or Sulfated Oils D5565 - 95(2011)Standard Test Method for Determination of the Solidification Point ofFatty Acids Contained in Animal, Marine, and Vegetable Fats and OilsD5566 - 95(2011) Standard Test Method for Determination of InorganicSalt Content of Sulfated and Sulfonated Oils

TABLE 7 ASTM's Leather Standards-Footwear Designation Test Title D2098 -13 Standard Test Method for Dynamic Water Resistance of Shoe UpperLeather by the Dow Corning Leather Tester D2099 - 14 Standard TestMethod for Dynamic Water Resistance of Shoe Upper Leather by the MaeserWater Penetration Tester D2210 - 13 Standard Test Method for Grain Crackand Extension of Leather by the Mullen Test D2322 - 14 Standard TestMethod for Resistance of Shoe Upper Leather to Artificial PerspirationD2346 - 13 Standard Test Method for Apparent Density of Leather D2941 -13 Standard Test Method for Measuring Break Pattern of Leather (BreakScale) D6015 - 14 Standard Test Method for Static Water Absorption ofLeather D7340 - 07(2012)e1 Standard Practice for Thermal Conductivity ofLeather

TABLE 8 ASTM's Leather Standards-Physical Properties Designation TestTitle D1516 - 05(2010) Standard Test Method for Width of Leather D1610 -01(2013) Standard Practice for Conditioning Leather and Leather Productsfor Testing D1813 - 13 Standard Test Method for Measuring Thickness ofLeather Test Specimens D1814 - 70(2015) Standard Test Method forMeasuring Thickness of Leather Units D1815 - 00(2015) Standard TestMethod for Water Absorption (Static) of Vegetable Tanned Leather D2207 -00(2015) Standard Test Method for Bursting Strength of Leather by theBall Method D2209 - 00(2015) Standard Test Method for Tensile Strengthof Leather D2211 - 00(2015) Standard Test Method for Elongation ofLeather D2212 - 00(2015) Standard Test Method for Slit Tear Resistanceof Leather D2347 - 00(2015) Standard Test Method for Measuring Area ofLeather Test Specimens D2813 - 03(2013) Standard Practice for SamplingLeather for Physical and Chemical Tests D4704 - 13 Standard Test Methodfor Tearing Strength, Tongue Tear of Leather D4705 - 13 Standard TestMethod for Stitch Tear Strength of Leather, Double Hole D5052 - 00(2010)Standard Test Method for Permeability of Leather to Water Vapor D6076 -08(2013) Standard Test Method for Shrinkage Temperature of LeatherD6182 - 00(2015) Standard Test Method for Flexibility and Adhesion ofFinish on Leather D6183 - 00(2015) Standard Test Method for Tackiness ofFinish on Leather D7255 - 14 Standard Test Method for AbrasionResistance of Leather (Rotary Platform, Abraser Method)

TABLE 9 ASTM's Leather Standards-Upholstery Designation Test TitleD1912 - 00(2010) Standard Test Method for Cold-Crack Resistance ofUpholstery Leather D2097 - 03(2010) Standard Test Method for FlexTesting of Finish on Upholstery Leather D2208 - 00(2010) Standard TestMethod for Breaking Strength of Leather by the Grab Method D6077 - 10Standard Test Method for Trapezoid Tearing Strength of Leather D6116 -00(2010) Standard Test Method for Blocking D7912 - 14 Standard TestMethod for Resistance of Finish to Heat Aging (Finish Stability)

TABLE 10 ASTM's Leather Standards-Vegetable Leather Designation TitleD1611 - 12 Standard Test Method for Corrosion Produced by Leather inContact with Metal D2213 - 00(2010) Standard Test Method forCompressibility of Leather D2875 - 00(2010) Standard Test Method forInsoluble Ash of Vegetable-Tanned Leather D2876 - 00(2010) Standard TestMethod for Water-Soluble Matter of Vegetable-Tanned Leather D4786 -00(2010) Standard Test Method for Stitch Tear Strength, Single HoleD4831 - 00(2010) Standard Test Method for Buckle Tear Strength ofLeather D4899 - 99(2009) Standard Practice for Analysis of VegetableTanning Materials-General D4900 - 99(2009) Standard Test Method forLignosulfonates (Sulfite Cellulose) in Tanning Extracts D4901 - 99(2009)Standard Practice for Preparation of Solution of Liquid Vegetable TanninExtracts D4902 - 99(2009) Standard Test Method for Evaporation andDrying of Analytical Solutions D4903 - 99(2009) Standard Test Method forTotal Solids and Water in Vegetable Tanning Material Extracts D4904 -99(2009) Standard Practice for Preparation of Solution of LiquidVegetable Tannin Extracts D4905 - 99(2009) Standard Practice forPreparation of Solution of Solid, Pasty and Powdered Vegetable TanninExtracts D6020 - 00(2010) Standard Practice for Calculation of(Non-Mineral) Combined Tanning Agents and Degree of Tannage D6075 - 13Standard Test Method for Cracking Resistance of Leather D6401 - 99(2009)Standard Test Method for Determining Non-Tannins and Tannin in Extractsof Vegetable Tanning Materials D6402 - 99(2014) Standard Test Method forDetermining Soluble Solids and Insolubles in Extracts of VegetableTanning Materials D6403 - 99(2014) Standard Test Method for DeterminingMoisture in Raw and Spent Materials D6404 - 99(2014) Standard Practicefor Sampling Vegetable Materials Containing Tannin D6405 - 99(2014)Standard Practice for Extraction of Tannins from Raw and Spent MaterialsD6406 - 99(2014) Standard Test Method for Analysis of Sugar in VegetableTanning Materials D6407 - 99(2014) Standard Test Method for Analysis ofIron and Copper in Vegetable Tanning Materials D6408 - 99(2014) StandardTest Method for Analysis of Tannery Liquors D6409 - 99(2014) StandardPractice for Color Tests with Sheepskin Skiver D6410 - 99(2014) StandardTest Method for Determining Acidity of Vegetable Tanning Liquors

TABLE 11 ASTM's Leather Standards-Wet Blue Designation Title D4576 -08(2013) Standard Test Method for Mold Growth Resistance of Wet BlueD6656 - 14b Standard Test Method for Determination of Chromic Oxide inWet Blue (Perchloric Acid Oxidation) D6657 - 14ae1 Standard Test Methodfor pH of Wet Blue D6658 - 08(2013) Standard Test Method for VolatileMatter (Moisture) of Wet Blue by Oven Drying D6659 - 10(2015) StandardPractice for Sampling and Preparation of Wet Blue for Physical andChemical Tests D6714 - 01(2015) Standard Test Method for Chromic Oxidein Ashed Wet Blue (Perchloric Acid Oxidation) D6715 - 13 StandardPractice for Sampling and Preparation of Fresh or Salt-Preserved (Cured)Hides and Skins for Chemical and Physical Tests D6716 - 08(2013)Standard Test Method for Total Ash in Wet Blue or Wet White D7476 -08(2013) Standard Test Method for Brine Saturation Value of Cured(Salt-Preserved) Hides and Skins D7477 - 08(2013) Standard Test Methodfor Determining the Area Stability of Wet Blue Submersed in BoilingWater D7584 - 10(2015) Standard Test Method for Evaluating theResistance of the Surface of Wet Blue to the Growth of Fungi in anEnvironmental Chamber D7674 - 14a Standard Test Method forHexane/Petroleum Ether Extract in Wet Blue and Wet White D7816 - 12Standard Test Method for Enumeration of Halophilic and ProteolyticBacteria in Raceway Brine, Brine-Cured Hides and Skins D7817 - 12Standard Test Method for Enumeration of Yeast and Mold in Raceway Brine,Brine-Cured Hides and Skins D7818 - 12 Standard Test Method forEnumeration of Proteolytic Bacteria in Fresh (Uncured) Hides and SkinsD7819 - 12 Standard Test Method for Enumeration of Yeast and Mold onFresh (Uncured) Hides and Skins

In some embodiments, leather products can have physical propertiessimilar to real leather. In some embodiments, a synthetic leatherdisclosed herein or a leather product made therefrom can tensilestrength as measured by ASTM D-2209-95 of at least about 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000 lbs/in². In some embodiments, asynthetic leather disclosed herein or a leather product made therefromcan tensile strength as measured by ASTM D-2209-95 of less than about5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200,1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400,350, 300, 250, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 lbs/in².

In some embodiments, a synthetic leather disclosed herein or a leatherproduct made therefrom can have a slit of at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100 lbs as measured by ASTM-D2212-94. In some embodiments, asynthetic leather disclosed herein or a leather product made therefromcan have a slit of less than about 200, 150, 100, 95, 90, 85, 80, 75,70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1lbs as measured by ASTM-D2212-94. In some embodiments, a syntheticleather disclosed herein or a leather product made therefrom can have astitch of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100 when measured in accordance withASTM-D4705-93. In some embodiments, a synthetic leather disclosed hereinor a leather product made therefrom can have a stitch of less than about200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46,45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, 1. In some embodiments, a synthetic leatherdisclosed herein or a leather product made therefrom can have the slitand stitch values are at least about 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100 lbs., when measured in accordance with their respectivetests. In some embodiments, a synthetic leather disclosed herein or aleather product made therefrom can have a Bally flex of at least about5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000,40000, 45000, 50,000, 55000, 60000, 65000, 70000, 80000 as measured byASTM D6182.

Multiple cell layers can be assembled to form a synthetic leather (e.g.,a full thickness skin equivalent). A synthetic leather can comprise atop part, a middle part and a bottom part. The top part can comprise anepidermal layer. For example, the top part can be a single layerepidermal layer. The middle part can comprise a basement membranesubstitute. In some cases, the middle part does not have a basementmembrane substitute. For example, the middle part can have a layer ofnegligible thickness. The bottom part can have one or more dermallayers. In some cases, the bottom part has a single dermal layer placedon a scaffold (e.g., silk). In some cases, the bottom part has multipledermal layers (e.g., up to 5 layers) without any scaffold. In somecases, the bottom part has multiple dermal layers stacked atop eachother and placed on a scaffold (e.g., silk).

Adhesiveness between epidermal and dermal layers can be strong enough toresist layer splitting. In some cases, the cells layers can be assembledby adhering on to a scaffold. Natural or synthetic adhesives can be usedfor the assembly. A natural adhesive can be fibrin glue, cold glues,animal glue (e.g., bone glue, fish glue, hide glue, hoof glue, rabbitskin glue, meat glue), blood albumen glue, casein glue, vegetable glues(e.g., starch, dextrin glues, Canada balsam, pine rosin based glue,cocconia, gum Arabic, postage stamp gum, latex, library paste, methylcellulose, mucilage, resorcinol resin, or urea-formaldehyde resin), orany combination thereof. A synthetic adhesive can be Acrylonitrile,Cyanoacrylate (e.g., n-buthyl-2-cyanoacrylate glue), Acrylic, Resorcinolglue, Epoxy resins, Epoxy putty, Ethylene-vinyl acetate, Phenolformaldehyde resin, Polyamide, Polyester resins, Polyethylene,Polypropylene, Polysulfides, Polyurethane, Polyvinyl acetate (includingwhite glue (e.g. Elmer's Glue) and yellow carpenter's glue (Aliphaticresin), Polyvinyl alcohol, Polyvinyl chloride (PVC), Polyvinyl chlorideemulsion (PVCE), Polyvinylpyrrolidone Rubber cement, Silicones, andStyrene acrylic copolymer. For example, the assembly can be performedusing fibrin glue. For example, the assembly can be performed usingn-buthyl-2-cyanoacrylate glue.

In some cases, cell layers (e.g., substantially planar layers) arestacked to form a synthetic leather. A cell layer can have anorientation defined by the placement, pattern, or orientation ofmulticellular bodies. In some cases, each layer can be stacked with aparticular orientation relative to the support substrate and/or one ormore other layers. For example, one or more layers can be stacked withan orientation that includes rotation relative to the support substrateand/or the layer below, wherein the rotation can be between 0.1 and 180degrees, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, and 180 degrees, or incrementstherein. In other cases, all layers are oriented substantiallysimilarly.

Once stacking of the layers is complete, the layers in thethree-dimensional construct can be allowed to fuse to one another toproduce a synthetic leather. In some cases, the layers fuse in a cellculture environment (e.g., a Petri dish, cell culture flask, bioreactor,etc.). In some cases, the fusing take place over about 15, 20, 25, 30,35, 40, 45, 50, 55, and 60 minutes, and increments therein. In othercases, fusing takes place over between 1 and 48 hours, e.g., over about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, and 48 hours, and incrementstherein. For example, fusing can take place over about 2 hours to about24 hours.

Culturing Condition

The cells and cell layers can be cultured in various cell cultureconditions. The cells or cell layers can be cultured in vitro. Forexample, a dermal layer and/or an epidermal layer can be cultured invitro. Alternatively, the cells or cell layers can be cultured in vivo.For example, a dermal layer and/or an epidermal layer can be cultured invivo.

The cells and cell layers can be cultured with one or more supplements.The one or more supplements can be natural supplements, syntheticsupplements, or a combination thereof. In some cases, a supplement canbe an additive. In some cases, one or more of the supplements induceproduction and assembly of extracellular matrix from iPSC-derivedfibroblasts, thus enhancing natural look of the synthetic leather.Exemplary supplements can include ECM components such as collagen andfibrin, growth factors, small molecules such as ascorbic acid or thelike, macromolecules such as dextran sulphate, carrageenan, or the like.

The cell layers can be cultured with certain air humidity. For example,the cell layers (e.g., dermal layers or epidermal layers) can becultured at from about 20% to about 100% humidity. For example, thehumidity can be from about 40% to about 100%, from about 50% to about95%, from about 45% to about 90%, from about 55% to about 95%, fromabout 60% to about 90%, from about 70% to about 80%, from about 71% toabout 79%, from about 72% to about 78%, from about 73% to about 77%,from about 74% to about 76%, from about 60% to about 70%, from about 65%to about 75%, from about 70% to about 80%, from about 75% to about 85%,from about 80% to about 90%, from about 85% to about 95%, or from about90% to about 100%, from about 40% to about 60%, from about 45% to about55%, from about 46% to about 54%, from about 47% to about 53%, fromabout 48% to about 52%, from about 48% to about 53%, from about 49% toabout 54%, or from about 47% to about 51%.

Leather Processing

Tanning

Methods herein can comprise tanning at least a portion of a syntheticleather, e.g., at least a portion of a dermal layer and/or an epidermallayer in the synthetic leather. Tanning can make a synthetic leatherresemble a natural leather, which can be a durable and flexible materialcreated by the tanning of animal rawhide and skin, often cattle hide.Tanning herein can refer to the process of treating the skins of animalsto produce leather. Tanning can be performed various ways, includingvegetable tanning (e.g., using tannin), chrome tanning (chromium saltsincluding chromium sulfate), aldehyde tanning (using glutaraldehyde oroxazolidine compounds), syntans (synthetic tannins, using aromaticpolymers), bacterial dyeing, and the like.

Tanning can be performed to convert proteins in the hide/skin into astable material that will not putrefy, while allowing the material toremain flexible. Chromium can be used as tanning material. The pH of thecell layer or layered structure can be adjusted (e.g., lowered; e.g. topH about 2.8-3.2) to enhance the tanning; following tanning the pH canbe raised (“basification” to a slightly higher level, e.g., pH about3.8-4.2).

Tanning can be performed on cell layers, e.g., dermal layers andepidermal layers. Tanning can also be performed on layered structures,e.g., layered structures comprising at least a dermal layer and at leastan epidermal layer. In certain cases, tanning can also be performed on asynthesized leather. For example, tanning can be performed after formingcell layers, e.g., dermal layers or epidermal layers. For example,tanning can be performed after forming layered structures.

Tanning can be performed by modify the extracellular matrix (ECM)material. Tanning can be performed by modifying collagen in the ECM. Thetanning can be performed using a tanning agent, e.g., chromium(III)sulfate ([Cr(H₂O)₆]2(SO4)₃). Chromium(III) sulfate can dissolve to givethe hexaaquachromium(III) cation, [Cr(H₂O)₆]³⁺, which at higher pHundergoes processes called olation to give polychromium(III) compoundsthat are active in tanning, being the cross-linking of the collagensubunits. Some ligands include the sulfate anion, the collagen'scarboxyl groups, amine groups from the side chains of the amino acids,as well as masking agents. Masking agents can be carboxylic acids, suchas acetic acid, used to suppress formation of polychromium(III) chains.Masking agents can allow the tanner to further increase the pH toincrease collagen's reactivity without inhibiting the penetration of thechromium(III) complexes. Tanning can increase the spacing betweenprotein chains in collagen (e.g., from 10 to 17 Å), consistent withcross-linking by polychromium species, of the sort arising from olationand oxolation. The chromium can be cross-linked to the collagen.Chromium-tanned leather can contain between about 4% and 5% of chromium.This efficiency can be characterized by its increased hydrothermalstability of the leather, and its resistance to shrinkage in heatedwater. Other tanning agents can be used to tan the layered body andmodify the collagen.

Tanning can also be performed using other minerals. In some cases,tanning can be performed using agent based on alum, zirconium, titanium,iron salts, or a combination thereof,

Further Processing

Cell layers, layered structures, and synthetic leathers made herein canbe further processed after tanning. In some cases, methods providedherein further comprise one or more leather processing steps (e.g.,those used in traditional leather formation). Examples of processingsteps include: preserving, soaking, liming, unhairing, fleshing,splitting, deliming, reliming, bating, degreasing, frizing, bleaching,colouring, pickling, depickling, tanning, re-tanning (e.g., if color islost during processing), thinning, retanning, lubricating, crusting,wetting, sammying, shaving, rechroming, neutralizing, dyeing,fatliquoring, filling, stripping, stuffing, whitening, fixating,setting, drying, conditioning, milling (e.g., dry milling), staking,buffing, finishing, oiling, brushing, padding, impregnating, spraying,roller coating, curtain coating, polishing, plating, embossing, ironing,glazing, and tumbling.

The synthetic leather can be shaped by, for example, controlling thenumber, size, and arrangement of the multicellular bodies and/or thelayers used to construct the animal skin, hide, or leather. In othercases, the animal skin, hide, or leather can be shaped by, for example,cutting, pressing, molding, or stamping. The shape the synthetic leathercan be made to resemble a traditional animal skin, hide, or leatherproduct.

Methods herein can comprise removing a portion of a synthetic leatherproduced herein. In some cases, the method comprises removing at least aportion of epidermal layer to form a removed product. For example, theremoving can be shaving.

Pigmentation

Methods herein can comprise pigmenting the synthetic leather. In somecases, pigmentation can be performed by introducing pigments producingcells (e.g., melanocytes) in the synthetic leather. In some cases, thesynthetic leather comprises functional live melanocytes. The melanocytescan have a similar location to that in the human skin. In some cases,melanin can be constitutively produced by melanocytes. In some cases,melanin can be transferred to keratinocytes. In some cases, melanocytesare produced upon stimulation, e.g., UV radiation or by propigmentingactive agents, such as alpha melanocyte stimulating hormone (aMSH),endothelin 1 (ET1), stem cell factor (SCF), prostaglandins E2 and F2α(PGE2, PGF2α), basic fibroblast growth factor (bFGF) or nerve growthfactor (NGF).

Differentiation of Progenitor Cells to Cells in a Synthetic Leather

Cells in epidermal layers, such as keratinocytes and melanocytes, aswell as cells in dermal layers, such as fibroblasts can be derived,e.g., differentiated, from progenitor cells, such as iPSCs. In othercase, primary cells or cultured cells derived from primary cells can beused to form cell layers to make synthetic leather.

Various methods of differencing iPSCs to cells in a synthetic leather,e.g., keratinocytes, melanocytes, or fibroblasts can be used. In somecases, differentiation of iPSCs to keratinocytes and building 3Depidermis from the iPSC-derived keratinocytes can be performed usingmethod described by Petrova et al., 3D In vitro model of a functionalepidermal permeability barrier from human embryonic stem cells andinduced pluripotent stem cells. Stem Cell Reports. 2014 Apr. 24;2(5):675-89. In other cases, cell layers can be formed using primarycells. For example, building 3D epidermis from primary keratinocytes canbe performed using the method described in Sun R et al., Loweredhumidity produces human epidermal equivalents with enhanced barrierproperties. Tissue Eng Part C Methods. 2015 January; 21(1):15-22.

In some cases, the methods described herein provide high-throughputmethods that reliably, accurately, and reproducibly scale up tocommercial levels the production of synthetic leather. Advantages of thesynthetic leather, engineered epidermal equivalent, engineered fullthickness skin equivalent and methods of making the same disclosedherein include, but are not limited to, production of customized tissuesin a reproducible, high throughput and easily scalable fashion withappealing appearance, texture, thickness, and durability. In someembodiments, the methods described herein can produce increase yields ofone or more of an epidermal layer, dermal layer, layered structure orsynthetic leather. In some embodiments, increase yields can be at leastabout 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or about 15 times yield compared to a comparable method.In some embodiments, the methods disclosed herein can reduce the cost ofthe manufacture of synthetic leathers, artificial epidermal layers,artificial dermal layers, layered structures, and products producedtherefrom. In some embodiments, the methods disclosed herein can produceuniform thickness synthetic leathers, artificial epidermal layers,artificial dermal layers, layered structures, and products producedtherefrom. In some embodiments, the synthetic leathers, artificialepidermal layers, artificial dermal layers, layered structures, andproducts produced therefrom can have a substantially uniform thickness,length and/or width. In some embodiments, cells in any one or more ofthe epidermal layers, dermal layers, layered structures can behomogeneously distributed. In some embodiments, cells in any one or moreof the epidermal layers, dermal layers, layered structures can beheterogeneously distributed.

Comparative Analysis of Epidermal Equivalent

FIG. 4A-4C. Illustrates a Comparative analysis of fine leather, nativeskin and epidermal equivalent. FIG. 4A illustrates FESEM of longitudinalsections of native skin and fine leather. FIG. 4A show distinctmorphological structures of epidermis (e) and dermis (a′) Tanningpermanently altered the structure of the skin. Borders between theindividual cells in epidermis became indistinguishable. Removingmoisture caused collagen bundles in dermis to become more compact anddurable. Magnification: 1000×.

FIG. 4B depicts FESEM images. In one instance, FIG. 4B depicts that bothsurface of fine leather and surface of epidermal equivalents, have asimilar smooth appearance indicating that they will likely induce acomparable tactile (touch) experience. Magnification: 2000×.

FIG. 4C depicts FESEM of longitudinal sections of fine leather andepidermal equivalent. Before the tanning takes place, similar toepidermis of native skin FIG. 4A, individual cell layers aredistinguishable in epidermal equivalent. As collagen in the dermis canbe responsible largely for tensile strength of the skin, collagenbundles can give thickness and durability to leather (inset), but maynot give sensory experience, which may entirely rely on outer layers ofepidermis.

FIG. 5A-5C illustrate a comparative analysis of stratum corneum (SC;cornified layer) of native skin and epidermal equivalent. FIG. 5Adepicts FESEM images showing the surface of epidermal equivalents appearsmoother than the surface of native skin, which may be due to thecontrolled environment of cell culture. FIG. 5B illustrates using TEMcorneodesmosomes (arrows), the principal “mechanical” junctions of theSC, could be detected as electron denser areas in epidermal equivalents.FIG. 5C illustrates that corneodesmosin (CDSN) can be synthesized andexcreted into the extracellular spaces by cells in SG, shortly beforeonset of cornification. CDSN can embed within the intercellular portionsof the SG desmosomes occupied by cadherins and in such a way can formcorneodesmosomes. Arrows point to similarly aligned dotty accumulationsof CDSN in SC of native skin and epidermal equivalent, likelyrepresenting corenodesmosomes. SC, stratum corneum; SG, stratumgranulosum; SS, stratum spinosum.

FIG. 6A-6E illustrate a comparative analysis of stratum granulosum (SG;granular layer) of native skin and epidermal equivalent. FIG. 6Aillustrates that Loricrin (LOR) staining. LOR is a major proteincomponent of the cornified cell envelope and can be expressed in thegranular layer of keratinizing epithelia. A similar LOR expressionpattern (dark brown pigment on H&E-stained tissue sections pointed byarrows) in SG was detected in both epidermis of native skin (left sideof the panel) and epidermal equivalent (right side of the panel). d,dermis; H&E, hematoxylin & eosin; SB, stratum basale; SC, stratumcorneum; SG, stratum granulosum; SS, stratum spinosum; *, Transwellfilter membrane. In FIG. 6B, epidermal Ca⁺⁺ gradient can be captured ontransmission electron microscopy as electron-dense precipitates. Ca⁺⁺deposits were present in SG and absent from SC in both native skin invivo (left side of panel) and epidermal equivalents generated in vitro(right side of the panel). SG, stratum granulosum; SS, stratum spinosum.In FIG. 6C, permeability barrier integrity was assessed by lanthanumperfusion. Lanthanum was visualized as electron-dense deposits in theextracellular spaces of the viable SG, demonstrating that lanthanum and,by extension, water and other small ions can pass between keratinocytesin this stratum. In contrast, lanthanum cannot penetrate further intothe SC because a functioning lipid barrier is blocking its movementupward. Epidermal equivalent generated in vitro, demonstrated equallyfunctional permeability barrier as native skin. SG, stratum granulosum;SS, stratum spinosum. FIG. 6D illustrates that tight junction protein1/zonula occludens-1 (TJP1/ZO-1) anchors tight junction strand proteins,which are fibril-like structures within the lipid bilayer, to the actincytoskeleton. Arrows point to similarly aligned bright green cellmembrane-associated accumulations of TJP1/ZO-1 in SG of native skin invivo (left side of panel) and epidermal equivalents generated in vitro(right side of the panel). SC, stratum corneum; SG, stratum granulosum;SS, stratum spinosum. FIG. 6E illustrates that Filaggrin (FLG) monomers,tandemly clustered into a large, 350 kDa protein precursor known asprofilaggrin, are present in the keratohyalin granules in cells of theSG. Arrows point to similarly aligned bright red granule and cellmembrane-associated accumulations of FLG in SG of native skin in vivo(left side of panel) and epidermal equivalents, generated in vitro(right side of the panel). SC, stratum corneum; SG, stratum granulosum;SS, stratum spinosum.

FIG. 7A-7C illustrate lipid bilayer formation in native skin andepidermal equivalents assessed with TEM. In FIG. 7A white arrows pointto normal lipid secretion at the border of SC and SG in both native skinin vivo (left side of panel) and epidermal equivalents generated invitro (right side of the panel). In FIG. 7B, lamellar bodies (whitearrowheads) are seen in the SG of both native skin in vivo (left side ofpanel) and epidermal equivalents generated in vitro (right side of thepanel). FIG. 7C depicts normal lipid bilayer (LB) morphology of nativeskin in vivo (left side of panel). Lipid bilayers in epidermalequivalents, generated in vitro (right side of the panel), had a similarappearance. SC, stratum corneum.

FIG. 8A-8D illustrate comparative analysis of markers of suprabasallayers of native skin and epidermal equivalent. FIG. 8A, FIG. 8B andFIG. 8C Keratin 10 (KRT10), keratin 1 (KRT1), desmocollin 1 (DCL1),markers of suprabasal layers, stratum spinosum (SS) and stratumgranulosum (SG), have a similar expression pattern in native skin invivo (left side of panel) and epidermal equivalents, generated in vitro(right side of the panel) as demonstrated by immunohistochemistry(KRT10; dark brown pigment on H&E-stained tissue sections pointed byarrows) and immunofluorescence (red cytoplasmic/KRT1/and red cellmembrane/DCL1/staining indicated by arrows). d, dermis; H&E, hematoxylin& eosin; SB, stratum basale; SC, stratum corneum; SG, stratumgranulosum; SS, stratum spinosum; * , Transwell filter membrane. In FIG.8D Desmosomes (arrows) are clearly defined in both native skin in vivoand epidermal equivalents generated in vitro. SS, stratum spinosum.

FIG. 9A-9D illustrate a comparative analysis of stratum basale (SB;basal layer) of native skin and epidermal equivalent. With regard toFIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D, MKI67, a marker ofproliferation, keratin 14 (KRT14), and transcription factor TP63 showtypical basal layer distribution in both native skin in vivo (left sideof panel) and epidermal equivalents generated in vitro (right side ofthe panel), as demonstrated by immunohistochemistry (MKI67; dark brownpigment on H&E-stained tissue sections pointed by arrows) andimmunofluorescence (green cytoplasmic/KRT14/and whitenuclear/TP63/staining indicated by arrows). Hemidesmosomes (arrows) areclearly defined in both native skin in vivo and epidermal equivalentsgenerated in vitro. BM, basement membrane; Cy, cytoplasm; d, dermis;H&E, hematoxylin & eosin; SB, stratum basale; SS, stratum spinosum; TM,Transwell filter membrane.

FIG. 10A-10F illustrate comparative analysis of extracellular matrixcomponents of basement membrane. Basement membrane (BM) can be formedfrom condensed networks of extracellular matrix (ECM) proteins, whichcan provide an essential structural scaffold on dermal-epidermaljunction. Integrin β 1 regulates multiple epithelial cell functions byconnecting cells with the ECM and it can be crucial for maintenance ofBM at dermal-epidermal junction. In FIG. 10A, integrin β1 show typicalbasal layer distribution in both native skin in vivo (left side ofpanel) and epidermal equivalents generated in vitro (right side of thepanel) as indicated by arrows (cell membrane-associated staining) andarrowheads (on the tip of cells protruding through the holes onTranswell membrane). BM, basement membrane; d, dermis; SB, stratumbasale; SS, stratum spinosum; SG, stratum granulosum; TM, Transwellmembrane.

Fibronectin can play a role in cellular adhesion. FIG. 10B illustatresthat in native skin in vivo (left side of panel) fibronectin can beexpressed mostly in dermis and relatively little can be detected in theBM area. Similarly, in epidermal equivalents generated in vitro (rightside of the panel), fibronectin can be detected on the tip of cellsprotruding through the holes on Transwell membrane (red arrowheads). d,dermis; SB, stratum basale; SS, stratum spinosum; SG, stratumgranulosum; TM, Transwell membrane.

The mechanical support provided by the BM can be determined by thecollagen IV or, in the case of epidermal equivalents, scaffold. In FIG.10C, As indicated with arrowheads, collagen IV expression has a similarpatchy pattern in native skin in vivo (left side of panel) as epidermalequivalents generated in vitro (right side of the panel). BM, basementmembrane; d, dermis; SB, stratum basale; SS, stratum spinosum; SG,stratum granulosum; TM, Transwell membrane.

Collagen VI can play a role in cellular adhesion, and can be associatedwith fibronectin. In native skin in vivo (left side of panel), collagenVI can expressed mostly in dermis and relatively little can be detectedin the BM area as in FIG. 10D. Similarly, in epidermal equivalentsgenerated in vitro (right side of the panel), collagen VI can bedetected on the tip of cells protruding through the holes in theTranswell membrane (arrowheads), FIG. 10D. BM, basement membrane; d,dermis; SB, stratum basale; SS, stratum spinosum; SG, stratumgranulosum; TM, Transwell membrane.

Collagen VII can anchors basement membrane for collagen I and IIIfibrils in dermis. In FIG. 10E, as pointed with arrowheads, collagen VIIexpression has a similar patchy pattern in native skin in vivo (leftside of panel) as epidermal equivalents generated in vitro (right sideof the panel). BM, basement membrane; d, dermis; SB, stratum basale; SS,stratum spinosum; SG, stratum granulosum; TM, Transwell membrane.

Laminin 5 (chain composition α3β3γ2) can be a major component ofanchoring filaments and can be essential for the initial assembly of theBM in vivo. In FIG. 10F as indicated with arrowheads, Laminin 5expression has a similar pattern in native skin in vivo (left side ofpanel) as epidermal equivalents generated in vitro (right side of thepanel). In addition to its extra abundance in epidermal equivalents,traces of laminin 5 can be seen on cell membrane of basal layer cells(arrows). BM, basement membrane; d, dermis; SB, stratum basale; SS,stratum spinosum; SG, stratum granulosum; TM, Transwell membrane.

FIGS. 11A-11I illustrate a structural analysis of full-thickness skinequivalent (FSE). FIGS. 11A and 11B depict cross sections of FSEdisplays distinct cellular layers of epidermis under 2600× magnification(FIG. 11A) and 5200× magnification (FIG. 11B). FIG. 11C depicts asurface of an FSE at 900× magnification having a similar smoothappearance as fine leather, indicating that an FSE can induce acomparable tactile (touch) experience. FIGS. 11D-1F depict longitudinalsections of dermal scaffold with residing dermal fibroblasts and richextracellular matrix at 91× magnification (FIG. 11D), 162× magnification(FIG. 11E) and 405× magnification (FIG. 11F). FIGS. 11G-11I depictdermal scaffolds with residing dermal fibroblasts and rich extracellularmatrix at 80× magnification (FIG. 11G), 695× magnification (FIG. 11H)and 2700× magnification (FIG. 11I).

FIGS. 12A-12R illustrate a time-course of engineering dermal equivalent.FIGS. 12A-12I depict day 2 after seeding dermal fibroblasts ontoscaffold at 36× magnification (FIG. 12A), 695× magnification (FIG. 12B),1470× magnification (FIG. 12C), 7750× magnification (FIG. 12D), 2320×magnification (FIG. 12E), 2420× magnification (FIG. 12F), 6560×magnification (FIG. 12G), 17000× magnification (FIG. 12H) and 22000×magnification (FIG. 12I). Cells can begin migrating into hollowstructures of a scaffold and secreting extracellular matrix. FIGS.12J-12 R depict day 7 after seeding dermal fibroblasts onto scaffold at64× magnification (FIG. 12J), 100× magnification (FIG. 12K), 364×magnification (FIG. 12L), 82× magnification (FIG. 12M), 253×magnification (FIG. 12N), 3940× magnification (FIG. 12O), 5550×magnification (FIG. 12P), 9440× magnification (FIG. 12Q) and 21680magnification (FIG. 12R). Longitudinal (FIGS. 12J-12L) and transversalsections (FIGS. 12M-12R) can show denser cells and richer extracellularmatrix, with some areas having almost complete obstruction of the hollowstructure of the scaffold (FIGS. 12M-12P).

EXAMPLES Example 1. Differentiation of iPSCs to Keratinocytes

To induce differentiation, undifferentiated iPSCs are transferred into a20% O₂ atmosphere environment and treated with mTESR1 or otherpluripotent stem cell basal media supplemented with 1 mM ATRA(Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction).

To select for cells with early acquisition of ectodermal fate, the cellsare harvested and replated onto freshly prepared 3D HDF ECM or othertype of ECM at a density of 5˜10×10³ cells per cm² and grown inDulbecco's modified Eagle's medium/Ham F12 (3:1; Life Technologies) orKeratinocyte media supplemented with serum substitute such as humanplatelets lyste and with 1 mM ATRA and 25 ng/ml BMP4 for a further 7days (Selection).

To enrich for putative epidermal progenitors, rapid adhesion to type IVcollagen-coated dishes can be used, and the rapidly adhering cells arecultured in defined keratinocyte-SFM or other keratinocyte mediumsupplemented with 1 mM ATRA for 7 days (Enrichment). After that, thecells are cultured in EpiLife medium (Life Technologies) or otherkeratinocyte medium for a further 7 days (Expansion) before finalharvest and analysis.

Example 2. Differentiation of Induced Pluripotent Stem Cells into aKeratinocyte Lineage

Coating Tissue Culture Dishes with Geltrex and Col

The procedure may be performed in a biological safety cabinet usingaseptic techniques. Similar to Matrigel, Geltrex matrix solidifiesrapidly at room temperature (RT). Aliquot each new batch of the matrixupon arrival and use pre-chilled pipet tips, racks and tubes whileworking with the reagent. 50, 100 and 200 μL aliquots are made andstored at −80° C. Use Geltrex at 1:100 dilutions.

The coating procedure below can be described for a 60 mm tissue culturedish. If a larger dish is to be used, adjust the volume of the coatingsolution accordingly. 1. Remove a 50 μL aliquot of Geltrex from the −80°C. freezer, and place it on ice in the biological safety cabinet. 2. Add5 mL of cold sterile DMEM/F12 to a 15 mL conical tube. 3. Use a 1 mLglass pipet, take 1 mL cold DMEM/F12 from the 15 mL conical tubeprepared in step 2, and add to the frozen Geltrex. Gently pipet up anddown to thaw and dissolve Geltrex. Transfer the dissolved Geltrex to therest of DMEM/F12 in the 15 mL conical tube prepared in step 2. Pipet tomix diluted Geltrex. 4. Add 50 μL of 3 mg/mL ColI stock solution intodiluted Geltrex from step 3. Pipet to mix diluted Geletrex with ColI.Add 4 mL of coating solution into 60 mm dish. Tap or swirl the plate toensure that the entire surface is coated. 5. Incubate the dish withGeltrex/ColI coating solution at 37° C. in the tissue culture incubatorfor at least 1 h. 6. Once the coating is complete, leave the coatingsolution in the dish and proceed with the plating of iPSCs as describedin the next subsection. Alternatively, aspirate the coating solution andadd 2 mL of fresh DMEM/F12 into the coated dish to prevent it fromdrying before plating the cells.

Plating iPSCs for Differentiation

Prepare one 60 mm tissue culture dish of feeder-free iPSCs grown to ˜70%of confluency. Examine cells under a microscope to confirm the absenceof contamination and the maintenance of their undifferentiatedphenotype. If the cells are stressed or dying, they start todifferentiate, presenting themselves as “cobblestone” areas with largerpolymorphic cells, and should not be used for the differentiation towardkeratinocytes.

For iPSC differentiation toward keratinocytes, a 1:8 split ratio ofiPSCs. 1. Prewarm N2B27 medium and Dispase in the 37° C. water bath. 2.Using the microscope, confirm that the colonies are ready for passaging.Gently aspirate medium from the dish. Add 2 mL of 1×PBS, swirl the plateto wash the cells, and gently aspirate PBS. 3. Add 1 mL of Dispase andreturn the plate to the 37° C. tissue culture incubator for 3-5 min. 4.While the cells are being incubated with Dispase, gently aspirate theGeltrex/ColI coating solution (or DMEM/F12) from step 6 in theGeltrex/ColI coating procedure and add 4 mL of complete N2B27 mediuminto the coated dish. 5. After 3˜5 min incubating with Dispase, confirmthat the cells are ready to be picked by looking for rolled or foldededges around the colonies. 6. Transfer the plate to the biologicalsafety cabinet and carefully aspirate Dispase. After the treatment withDispase, the colonies are very loosely attached to the surface of thedish and may peel off if too much force is used. 7. Gently add 2 mL ofplain DMEM/F12. Aspirate off the medium and repeat the wash 3 times. 8.Add 2 mL of complete N2B27 into the dish and gently scrape the coloniesoff the plate. Transfer the cells from the dish into a 15 mL conicaltube and add 6 mL of complete N2B27 bring the total volume of cellsuspension to 8 mL. 9. Gently mix the cell suspension to break largeclumps of cells. Transfer 1 mL of the cell suspension to the coated dishprepared in step 3 of the current subsection. Discard or replate theleftover cells using the conditions established for a given laboratory.10. Transfer the newly plated cells to the incubator and gently shakethe plate back and forth and side to side to distribute the cellsevenly. Incubate the cells overnight in the 37° C. tissue cultureincubator.

Differentiation of iPSCs with RA and BMP4

The differentiation and subculturing of iPSC-derived keratinocytes areto be performed in a biological safety cabinet using aseptic techniques.Examine the new plate the day after passaging to confirm the successfulattachment of iPSCs. If iPSCs start forming colonies, proceed with thedifferentiation protocol below.

1. Prewarm complete DKSFM (with antibiotics and DKSFM supplement) in the37° C. water bath.

2. Add 5 mL of prewarmed DKSFM from the previous step to a 15 mL conicaltube, add 5 μL of 1 mM RA to achieve 1 μM final working concentrationand 5 μL of 25 μg/mL BMP4 to achieve 25 ng/mL final workingconcentration, mix well. 3. Aspirate off N2B27 medium from the dish withplated iPSCs, wash once with 4 mL of 1×PBS, and add 4 mL of DKSFMcontaining 1 μM RA and 25 ng/mL BMP4 from the step above. This is day 1of differentiation procedure. 4. Transfer the cells to the incubator andincubate for 48 h. 5. Replace the medium with fresh DKSFM containing 1μM RA and 25 ng/mL BMP4 after 48 h of incubation. Transfer the cell tothe incubator for another 48 h. 6. After the second round of 48 hinduction (day 4 of differentiation), replace the medium with completeDKSFM without RA and BMP4. Incubate cells in the incubator for 10 daysin complete DKSFM, changing medium every other day. 7. On day 14 ofdifferentiation, prepare complete CnT-07 medium by adding antibioticsand provided supplements, pre-warm the medium. By this day, the majorityof the cells in the outgrown iPSC colony start exhibiting anepithelial-like phenotype. 8. Aspirate off DKSFM from differentiatedcells, and replace with 4 mL of complete CnT-07. Incubate the cells inthe tissue culture incubator for another 10 days, changing completeCnT-07 every other day.

Rapid Attachment and Culturing of iPSC-Derived Keratinocytes

On day 24 of differentiation, many cells that migrate away from theoutgrown iPSC colony exhibit a keratinocyte-like phenotype, and startexpressing p63, a master regulator required for the commitment of theectoderm to a keratinocyte fate, and Krt14. By this day, the 60 mm dishused for iPSC differentiation is fully confluent and need to bepassaged. To enrich for iPSC-derive keratinocytes during passaging, theof the differentiated iPSC culture is rapidly attached toColI/ColIVcoated plates. Up to four 100 mm ColI/ColIV-coated tissueculture dishes are used to perform the rapid attachment procedure fromone 60 mm dish containing differentiated iPSCs. If only one 100 mm dishis to be used, plate one fourth of the differentiated iPSC culture forthe rapid attachment procedure.

Coating Plates with ColI and ColIV

The procedure may be performed in the biological safety cabinet usingaseptic techniques. 1. Reconstitute ColIV powder to a concentration of 2mg/mL in sterile 0.25% Glacial acetic acid. Dissolve for several hoursat 2˜8° C., occasionally swirling. Make aliquots and store them at −20°C. 2. Thaw the aliquot of ColIV stock solution (2 mg/mL) very slowly byplacing the vial in an ice bucket and keeping it at 4° C. for severalhours. 3. Resuspend ColIV stock solution in the appropriate volume (5 mLper each 100 mm dish) of sterile 0.25% Glacial acetic acid to a finalworking concentration of 7 μg/mL. Add an appropriate volume of ColIstock solution to achieve a final working ColI concentration of 30μg/mL. Coat the plates by using 5 mL of working solution to cover a 100mm dish. Incubate the plates at room temperature in the biologicalsafety cabinet for 1 hour. 4. Aspirate the liquid from the coatedplates, rinse the dishes once with 5 mL of sterile 1×PBS and once with 5mL of ddH2O. 5. Air-dry the washed dishes in the biological safetycabinet. Use plates directly or seal them with Parafilm and store at 4°C. for up to 6 months. To use a previously stored ColIV-coated plate,allow the plate to warm up at room temperature in the biological safetycabinet for at least 1 hour prior to plating cells.

Rapid Attachment of iPSC-Derived Keratinocytes

1. On day 24 of differentiation, prewarm complete CnT-07, Accutase, andColI/ColIV-coated dish(es). 2. Wash the cells with 1×PBS, add 2 mL ofAccutase and incubate in the tissue culture incubator for 5 min. Confirmunder the microscope that cells start detaching. 3. Add 3 mL of completeCnt-07, pipet up and down to dislodge the cells and collect the cellsuspension into a 15 mL conical tube. Spin the cells down at 260×g for 5min and aspirate the supernatant. Resuspend the pellet in 10 mL ofcomplete Cnt-07 medium, repeat the spin at 260×g for 5 min, and aspiratethe supernatant. 4. Resuspend the pellet in 4 mL of complete CnT-07,pipet up and down to break cell clumps into single cells. 5. Add 9 mL ofcomplete CnT-07 medium into each ColI/ColIV-coated dish and transfer 1mL of cell suspension from step 4 above into each ColI/ColIV-coateddish. Allow the cells to attach to the coated dish at room temperaturefor 15-30 min. 6. Carefully aspirate the medium with the floating cells(these are undifferentiated or partially differentiated iPSCs). Do notdisturb the attached cells (these are iPSC derived Krt14 positivecells). Add 10 mL of fresh complete CnT-07 medium into the plate withthe attached cells. Let the cells expand in the 37° C. tissue cultureincubator, changing the medium every other day. Passage cells as neededwith Accutase in CnT-07 or EpiLife (with EDGS supplement) on ColI-coateddishes. After passage 2 or 3 and following the rapid attachment step,the culture should consist of ˜90% of Krt14 positive cells exhibiting akeratinocyte-like phenotype. The keratinocyte-like phenotype of theobtained culture can be verified by standard immunflorescence analysesfor Krt14 expression and by the ability to reconstitute a normalstratified epidermis in organotypic cultures.

Example 3. Preparing Epidermal Layer from Primary Keratinocytes

Primary keratinocytes are isolated from a single neonatal foreskin andgrown in 0.07 mM Ca²⁺154 CF medium (Life Technologies) supplemented withman keratinocyte growth supplement. A suspension of first-passagekeratinocytes (2.21×10⁵/cm² insert) is seeded on Cellstart CTS (LifeTechnologies) (or other ECM substrate) coated PET, 0.4-mm inserts (EMDMillipore) in CnT-07 media (CELLnTEC) or CnT-Prime media (CELLnTEC)according to manufacturer's protocol.

Day 3 (D3) after seeding, the media are switched to CnT-02-3D (CELLnTEC)or CnT-3D Barrier (CELLnTEC). On day 4, the HEEs are air exposed byfeeding the bottom of the insert with CnT-02-3D or CnT-3D Barrier. Fromday 4 onward, HEEs are fed daily with CnT-02-3D or CnT-3D Barrier untilharvested. HEEs are grown in a humid (at 100% RH) or dry incubator (at50% RH) at 37° C. and 5% CO₂. A dial hydrometer (Fisher Scientific) isused to measure incubator humidity. Low incubator humidity is maintainedby removal of water pan.

To control for possible changes in osmolarity, media are refresheddaily. Significant changes in osmolarity are not detected using thisprotocol, as measured by a Micro Osmometer (Precision Systems).Twelve-well inserts are used for transepithelial electrical resistance(TEER) measurements, light microscopy, and electron microscopy, whilesix-well inserts are used for transepidermal water loss (TEWL)measurements and immunoblotting.

Example 4. Culturing an Epidermal Layer

Keratinocytes are seeded at a density of 2.0-2.5×10⁵ cells/cm² ofpolyethylene terephthalate (PET) membrane with 0.4 μm pore inserts (EMDMillipore; Cat. No.: MCHT12H48) in CnT-07 media (CELLnTEC) or CnT-Primemedia (CELLnTEC).

Day 3 (D3) after seeding, the media are switched to CnT-02-3D (CELLnTEC)or CnT-3D Barrier (CELLnTEC). On day 4, the cells air exposed by feedingthe bottom of the insert with CnT-02-3D CnT-3D Barrier. From Day 4onward, the epidermal layer is fed daily with CnT-02-3D or CnT-3DBarrier until harvested at Day 14.

Example 5. Preparing Support Substrate

To prepare a 2% agarose solution, 2 g of Ultrapure Low Melting Point(LMP) agarose is dissolved in 100 mL of ultrapure water/buffer solution(1:1, v/v). The buffer solution may be optionally PBS (Dulbecco'sphosphate buffered saline 1×) or HBSS (Hanks' balanced salt solution1×). The agarose solution may be placed in a beaker containing warmwater (over 80° C.) and held on the hot plate until the agarosedissolves completely. The agarose solution remains liquid as long as thetemperature is above 36° C. Below 36° C., a phase transition occurs, theviscosity increases, and finally the agarose forms a gel.

To prepare agarose support substrate, 10 mL of liquid 2% agarose(temperature >40° C.) may be deposited in a 10 cm diameter Petri dishand evenly spread to form a uniform layer. Agarose is allowed for form agel at 4° C. in a refrigerator.

Example 6. Producing a Synthetic Leather Comprising Fibroblasts,Keratinocytes, and Melanocytes

The outline of the protocol can be as follow: a) bringing fibroblastsand a solution of collagen into contact, then incubating for asufficient period of time to obtain a contracted collagen matrix inwhich the fibroblasts are distributed, constituting a dermis equivalent,b) seeding, with a mixture of keratinocytes and melanocytes, the dermisequivalent obtained in a), and immersion culture in a liquid medium, c)immersion of the entire culture (keratinocytes and melanocytes seeded onthe dermis equivalent) obtained in b), and continuation of the cultureat the air-liquid interface until a pluristratified epidermis equivalentcontaining melanocytes, on a dermis equivalent containing fibroblasts ina collagen matrix, constituting a skin equivalent, is obtained.

Step a) can be carried out with collagen type I, in particular of bovineorigin, or a mixture of collagens I and III (approximately 30% relativeto the final volume of the lattice) in homogeneous suspension.Advantageously, other constituents are added thereto, such as laminin(in particular, from 1% to 15% relative to the final volume), collagenIV (in particular, from 0.3% to 4.5% relative to the final volume)and/or entactin (in particular, from 0.05% to 1% relative to the finalvolume) so as to obtain a homogeneous suspension. The fibroblasts areobtained from skin. They are cultured in a suitable medium, and thensuspended before mixing with the suspension of collagen and growthfactors. The mixture is incubated for 1 to 6 days, preferably for 4 or 5days, at a temperature of approximately 37° C., generally from 36° C. to37.5° C. Advantageously, the mixture is incubated on a support whichdoes not allow adhesion thereof, in particular which prevents adhesionof the mixture to the edges of the support; such a support may inparticular be obtained by prior treatment of its surface, for example bycoating said surface with bovine albumin or serum. A collagen gel whichis contracted freely in several directions, while discharging thenutritive medium, and in which the fibroblasts are embedded, is thusobtained.

In order to carry out step b), use can be made of keratinocytesoriginating from skin, preferably from adult skin. The keratinocytes areamplified before seeding according to the technique of Rheinwald andGreen (Cell, vol. 6, 331-344, 1975) by culture on a feeder supportconstituted of 3T3 fibroblasts in a suitable medium known to thoseskilled in the art, in the presence of growth factors, in particular ofamino acids, serum, cholera toxin, insulin, triiodothyronine and pHbuffer solution. In particular, such a culture medium may especiallycontain at least one mitogenic growth factor for keratinocytes (forexample, epidermal growth factor (EGF) and/or keratinocyte growth factor(KGF), in particular KGF), insulin, hydrocortisone and, optionally, anantibiotic (for example: gentamycin, amphotericin B).

The melanocytes can be melanocytes originating from young or adultanimal skin. They are amplified by culture in a suitable medium, in theabsence of phorbol ester, composed of a base medium such as DMEM/F12 orMCDB153 and supplemented with melanocyte-specific growth factors (suchas, for example, bFGF, SCF, ET-1, ET3 or aMSH), and in particular in M2medium (Promocell) or in other media such as M254 (Cascades Biologics™).

Cell suspensions of melanocytes and of keratinocytes are prepared fromthese cultures, and mixed so as to obtain mixed keratinocyte/melanocytesuspensions. The melanocyte/keratinocyte ratio may be from 1:10 to 2:1,and is generally approximately 1:1. This mixed suspension is depositedon the dermis equivalent. The dermis equivalent is advantageouslyattached to a support via a biological material such as collagen. Themelanocyte/keratinocyte suspension is deposited in a ring or anyequivalent means for maintaining it on a delimited surface part. Aliquid nutritive medium is added in such a way as to cover the mixtureof cells. This medium contains growth factors known to those skilled inthe art, in particular EGF and/or KGF. The medium will be replacedregularly and the culture continued as an immersion, generally for aperiod of from 2 to 10 days, in particular from 5 to 8 days, andapproximately 7 days. The medium contains KGF starting from the 2nd dayof immersion, and ideally starting from the 4th day of immersion.

The skins are subsequently, in a manner known per se, immersed so as toobtain differentiation of the keratinocytes and formation of astratified epidermis equivalent. This step c) corresponding to theculture as an immersion at the air-liquid interface is continued until adifferentiated structure is obtained, in general approximately 7 days.However, step c) may be continued for a longer period of time, forexample for approximately 28 days, while at the same time conserving askin equivalent having the advantageous characteristics specified in theabove text. The nutritive culture medium will be refreshed regularly.The skin equivalent is subsequently removed so as to perform requiredtests.

Example 7. Induction of Follicle Formation in Cultured Skin Specimens

Expanded DP cells are mixed with cultured ORS cells, washed, andcarefully resuspended in 20 ml of sterile phosphate buffered saline(PBS, Sigma) at suitable cell densities. Cultured DP and ORS cells usedin each experiment are obtained from different donors, because thedifferent duration of culture for DP and ORS cells do not allowpreparation of the two cell types from the same donor. The cellsuspension is slowly injected into the dermis of cultured skin pieces 1day after establishing the culture.

Example 8. Culturing Hair Follicle Cell Populations

Hair follicles are obtained from the occipital region. Dermal papilla(DP) cells are prepared and cultured as described in Randall et al., Acomparison of the culture and growth of dermal papilla cells from hairfollicles from non-balding and balding (androgenetic alopecia) scalp. BrJ Dermatol 1996: 134: 437-444).

Briefly the DP of the hair follicles is isolated under a dissectingmicroscope and transferred individually to a 24-well tissue cultureplate (Sarstedt). Cell culture is performed in DMEM, supplemented with15% FCS (Sigma). After initiation of cell proliferation, cells arecultured to confluency and expanded for two passages. For isolation ofouter root sheath (ORS) cells, the middle part of the hair follicles,containing the bulge region, is excised and subjected to mildtrypsinization. At least cells of 10 hair follicles are used for eachculture. The obtained cells are washed twice in RPMI-1640 medium (Sigma)and subjected to cell culture in standard keratinocyte medium (Epilife,Sigma). Cells are harvested after 1 week of culture.

Example 9. Tanning Full Thickness Skin Equivalents

Full thickness skin equivalents are tanned by chrome tanning. The firststep is ice and sulfuric acid treatment. This opens up the tissue so itcan receive the chromium. The chromium is then added along withmagnesium oxide.

The process brings the pH level of the full thickness skin equivalentsdown to around 3. After chromium has worked through the full thicknessskin equivalents the tanning liquor is then introduced which brings thepH level up to around 4. This is followed by a warm water bath and thenroll pressing to remove excessive liquid. The final stage is then toapply a surface treatment if necessary and then dry the full thicknessskin equivalents while stretched out and then re-press when done.

Example 10. X-Tan Tanning Protocol

Full thinkness skin equivalents can be tanned using an X-tan procedure.Prior to tanning, a full thinkness skin equivalent was limed, whichcomprises the steps of soaking the skin equivalent, adding a substrate,adjusting the pH, and washing. The skin equivalent was then be de-limedby washing the skin equivalent, adding a pre-deliming buffer, delimingthe skin equivalent, and washing. The skin equivalent was then tanned bywetting back, adding a tanning substrate, adjusting the pH to a pHconducive for the tanning, performing cycles of fixation and fixation,and fat liquoring to obtain the tanned skin equivalent.

Example 11. Full Thickness Skin Equivalents

A type I collagen matrix (containing 0.5×10⁶ iPSC derived fibroblasts)can be deposited onto polyethylene terephthalate membranes (BDBiosciences), and allowed to polymerize. After incubation of thepolymerized matrix for about 7 days, 1×10⁶ iPSC-derived keratinocytesand 0.1×10⁶ iPSC-derived melanocytes can be seeded onto the matrix, andincubated for a further 7 days. The composite culture can be raised tothe air-liquid interface and fed from below to induce epidermaldifferentiation. Full thickness skin equivalents can harvested about 14days later and either snap frozen in LN2 or embedded in wax. For melaninquantification

Example 12. Immunostaining of Frozen Section

Fixation:

Tissues can be fixed in 3.8% paraformaldehyde/phosphate-buffered saline(PBS), pH 7.2-7.6 for 30 minutes. The samples can be washed three times5 minutes in PBS. The tissue samples can be infiltrated with a series ofsterile sucrose gradients (10% sucrose overnight, 15% sucrose for 6-8hours, 30% sucrose overnight and finally in 30% sucrose mixed 1:1 withoptimal cutting temperature (OCT) compound overnight) rotating on 4° C.The samples can be embedded in OCT and frozen in liquid nitrogen vapor.The cryo-blocks can be stored at −80° C.

Cutting Sections:

The day before cutting, the cryo-blocks can be transferred to −20° C.overnight. Sections (10 μm) can be prepared using a standard cryostat.The sections can be kept at −20° C. untill processing.

Processing:

Control incubations can be included. Preimmune sera or isotype-matchednonimmune antibodies can be used instead of the primary antibodies. Thesections can be submerged in either 90% cold acetone for 10 minutes or0.2% triton X-100/PBS for 5 minutes to expose antigens. The samples canbe washed three times 5 minutes in PBS. Nonspecific antibody reactivitywas blocked by submerging the sections into 5% BSA with 0.1% tritonX-100 for 1 hr. The sections can be then incubated overnight at 4° C.with a mixture of the two antibodies: i) 2.5 μg/ml of ChromPure donkeywhole IgG (for purpose of blocking; all secondary antibodies can be madein donkey); ii) 1 μg/ml of appropriate primary antibody. The sectionscan be rinsed three times 5 minutes in PBS. The sections can beincubated for 30 to 60 min at room temperature with the appropriatespecies-specific secondary antibody, made in donkey, and conjugated toeither red or green fluorophore. The sections can be washed three times5 minutes in PBS. The sections can be then incubated for 10 minutes with10 μg/ml Hoechst 33342 at room temperature. The sections can be washed 3times 5 minutes with PBS.

Visualization:

The samples can be mounted with Vectashield medium (Vector) and thesamples can be visualized with epifluorescence microscope (Zeiss),equipped with appropriate filters.

Example 13. Immunostaining of Paraffin Embedded Sections

Fixation:

Tissues can be fixed in 3.8% paraformaldehyde/phosphate-buffered saline(PBS), pH 7.2-7.6 for 30 minutes. The samples can be washed three times5 minutes in PBS. The tissue samples can be dehydrated in ascendingethanol series (50%, 70%, 2×100%; 20 min each) and clearing agent(xylene, 2×20 min). The samples can be perfused with paraffin wax at 65°C. 2×1 hour and embedded in paraffin blocks. The paraffin blocks can bestored at room temperature until further use.

Cutting Sections:

The tissue was sectioned at 5 μm thickness using a standard microtome.The sections can be kept at room temperature until processing.

Processing:

Control incubations can be included. Preimmune sera or isotype-matchednonimmune antibodies can be used instead of the primary antibodies. Thesections can be re-hydrated in ascending series xylene/ethanol series 2×xylene, 2×100% ethanol, and 1×70% and 50%; 10 min each. The sections canbe then briefly rinsed with tap water. The sections can be then stainedwith hematoxylin for 5 minutes. The sections can be then washed withdH₂O until solution was clear. The sections can be stained with 0.5%Eosin for 10 minutes. The sections can be then rinsed briefly in tapwater. Nonspecific antibody reactivity was blocked by submerging thesections into 5% BSA for 1 hr. The sections can be then incubated forovernight at 4° C. with a mixture of the two antibodies: i) 2.5 μg/ml ofChromPure donkey whole IgG (for purpose of blocking; all secondaryantibodies are made in donkey); ii) 1 μg/ml of appropriate primaryantibody. The sections can be rinsed three times 5 minutes in PBS. Thesections can be incubated for 30 min at room temperature with theappropriate species-specific secondary antibody, made in donkey, andconjugated to horseradish peroxidase (HRP). The sections can be washedthree times 5 minutes in PBS.

Visualization:

The samples can be incubated with 3, 3′-diaminobenzidine (DAB) substratekit (VectorLaboratories) according to manufacturer's protocol. DAB yielda brown stain. If nickel chloride is added to the substrate solution, agray-black stain can result. The samples can be dehydrated in ascendingethanol series (50%, 70%, 2×100%; 10 min each) and clearing agent(xylene, 2×10 min). The samples can be mounted in mounting medium andvisualized with phase contrast microscope (Zeiss) equipped with digitalcamera.

Example 14. Field Emission Scanning Electron Microscopy (FESEM)

Fixation:

Samples can be fixed for 24 hours at 4° C. with 4% paraformaldehyde and2% glutaraldehyde in 0.1M Sodium Cacodylate Buffer (pH7.4) and placed in0.1 M sodium cacodylate buffer and maintained at 4° C. prior to furtherprocessing.

Processing:

The samples can be post-fixed for 1 hour with 1% aqueous OsO4. Afterdehydration in an ascending ethanol series (50%, 70%, 2×100%; 10 mineach) samples can be critical point dried with liquid CO₂ in a TousimisAutosamdri-815B apparatus, mounted with double-sided copper tape onto 15mm aluminum mounts, and sputter-coated with 40 Å of gold-palladium usinga Denton Deskll Sputter Coater.

Visualization:

Cross sections of duplicate samples can be mounted onto low profile45/90 degree SEM mounts for analysis of internal morphology.Visualization can be performed with a Zeiss Sigma FESEM (Carl ZeissMicroscopy, Thornwood, N.Y.) operated at 2-3 kV, using inLens SecondaryElectron (SE) detection, as well as mixed signal InLens/SE2 (75/25%)detection at working distance 3-5 mm. Images can be captured in TIFFusing store resolution 2048×1536 and a line averaging noise reductionalgorithm.

Processing:

Previously dried samples (i.e. leather) can be cut to size andsputter-coated with 40 Å of gold-palladium using a Denton Deskll SputterCoater.

Visualization:

Cross sections of duplicate samples can be mounted onto low profile45/90 degree SEM mounts for analysis of internal morphology.Visualization was performed with a Zeiss Sigma FESEM (Carl ZeissMicroscopy, Thornwood, N.Y.) operated at 2-3 kV, using inLens SecondaryElectron (SE) detection, as well as mixed signal InLens/SE2 (75/25%)detection at working distance 3-5 mm. Images can be captured in TIFFusing store resolution 2048×1536 and a line averaging noise reductionalgorithm.

Example 15. Transmission Electron Microscopy (TEM)

Fixation:

Samples can be fixed 30 minutes at 4° C. in 2% glutaraldehyde and 2%paraformaldehyde with 0.06% calcium chloride in 0.1 M sodium cacodylatebuffer, pH 7.4. The samples can be then placed in 0.1 M sodiumcacodylate buffer and maintained at 4° C. prior to further processing.

Processing:

The samples can be then washed and placed in either 0.2% rutheniumtetroxide (for visualization of lipid bilayers) or 1.5% osmium tetroxidewith 1.5% potassium ferrocyanide, in 0.1 M sodium cacodylate, pH 7.4, atroom temperature in the dark for 45 minutes. After rinsing in buffer,the samples can be dehydrated in a graded ethanol series (50%, 70%,2×100%; 10 min each), and subsequently embedded in a low-viscosity Epoxyresin.

Visualization:

Semi-thin sections can be stained with 1% toluidine blue with 1% azureII in 1% borax solutions and viewed under phase contrast microscope(Zeiss). Ultrathin sections can be collected and stained withwater-saturated 3% uranyl acetate and/or contrasted in 2.5% lead citrateon uncoated nickel grids. Ultrathin sections can be viewed with a Zeiss10 A electron microscope operated at 60 kV. Images can be captured inTIFF.

Ion Capture Cytochemistry (Ca⁺⁺ Gradient):

Fixation:

For ultrastructural Ca⁺⁺ localization, the samples can be fixed in 2%paraformaldehyde, 2% glutaraldehyde, 0.09 M potassium oxalate,containing 0.04 M sucrose. Samples can be subsequently fixed overnightat 4° C.,

Processing:

The samples can be post-fixed in 1% osmium tetroxide containing 2%potassium pyroantimonate, pH 7.4 for 2 hrs at 4° C. in the dark. Tissuesamples then can be washed in alkalinized water (pH 10) and transferredto ethanol solutions (50%, 70%, 2×100%; 10 min each) for dehydration andembedding in a low-viscosity, Epoxy resin.

Visualization:

Ultrathin sections can be collected and stained with water-saturated 3%uranyl acetate and/or contrasted in 2.5% lead citrate on uncoated nickelgrids. Ultrathin sections can be viewed with a Zeiss 10 A electronmicroscope operated at 60 kV. Images can be captured in TIFF.

Lanthanum Perfusion:

Fixation:

The perfusion pathway was assessed in all subjects by immersion ofsamples in 4% lanthanum nitrate in 0.05 M Tris buffer containing 2%glutaraldehyde, 1% paraformaldehyde, pH 7.4, for 1 hour at roomtemperature.

Processing:

The samples can be washed and placed in 1.5% osmium tetroxide with 1.5%potassium ferrocyanide, in 0.1 M sodium cacodylate, pH 7.4, at roomtemperature in the dark for 45 minutes. After rinsing in cacodylatebuffer, the samples can be dehydrated in a graded ethanol series (50%,70%, 2×100%; 10 minutes each), and subsequently embedded in alow-viscosity, Epoxy resin.

Visualization:

Ultrathin sections can be collected and stained with water-saturated 3%uranyl acetate and/or contrasted in 2.5% lead citrate on uncoated nickelgrids. Ultrathin sections can be viewed with a Zeiss 10 A electronmicroscope operated at 60 kV. Images can be captured in TIFF.

While some embodiments have been shown and described herein, suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the disclosure provided herein. It should beunderstood that various alternatives to the embodiments described hereincan be employed.

1.-198. (canceled)
 199. A tanned synthetic leather, which prior totanning comprises: an artificial dermal layer comprising fibroblast,wherein said fibroblast is differentiated from an induced pluripotentstem cell.
 200. The tanned synthetic leather of claim 199, which priorto tanning, further comprises an artificial epidermal layer.
 201. Thetanned synthetic leather of claim 200, wherein said epidermal layerfurther comprises a keratinocyte.
 202. The tanned synthetic leather ofclaim 201, wherein said keratinocyte is differentiated from an inducedpluripotent stem cell.
 203. The tanned synthetic leather of claim 200,wherein said epidermal layer is upon said dermal layer thereby forming alayered structure.
 204. The tanned synthetic leather of claim 203,wherein said layered structure further comprises a melanocyte.
 205. Thetanned synthetic leather of claim 200, wherein said epidermal layerfurther comprises collagen.
 206. The tanned synthetic leather of claim199, wherein said dermal layer further comprises collagen.
 207. Thetanned synthetic leather of claim 199, wherein said dermal layer isformed upon a scaffold.
 208. The tanned synthetic leather of claim 207,wherein said scaffold comprises silk.
 209. The tanned synthetic leatherof claim 208, wherein said scaffold comprises a natural tissue adhesive.210. The tanned synthetic leather of claim 209, wherein said naturaltissue adhesive comprises fibrin glue.
 211. The tanned synthetic leatherof claim 203, wherein said synthetic leather further comprises abasement membrane substitute.
 212. The tanned synthetic leather of claim211, wherein said basement membrane substitute is between said epidermallayer and said dermal layer.
 213. The tanned synthetic leather of claim212, wherein said basement membrane substitute comprises a driedacellular amniotic membrane.
 214. The tanned synthetic leather of claim199, comprised in one or more of a watch strap, a belt, a packaging, ashoe, a boot, a footwear, a glove, a clothing, a luggage, a bag, aclutch, a purse, a backpack, a wallet, a saddle, a harness or a whip.